HIV integrase inhibitors for treatment of HIV infection

Abstract

The present invention relates to hydroxyl-substituted fused tetracyclic heterocycle compounds, including a compound of formula (1): and pharmaceutically acceptable salts thereof, which compounds exhibit activity against HIV integrase. The present invention also relates to compositions comprising these compounds, and methods of making these compounds. The invention further relates to methods of using these compounds for treating HIV infection in a subject, including administering these compounds once-weekly for treatment of HIV infection.

Description

HIV INTEGRASE INHIBITORS FOR TREATMENT OF HIV INFECTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U. S. Provisional Patent Application Serial No. 63 / 717,438 filed November 7, 2024. the entire contents of which are incorporated by reference herein.FIELD OF THE INVENTION

[0002] This disclosure relates to hydroxyl-substituted fused tricyclic heterocycle compounds that exhibit activity against HIV integrase, compositions comprising these compounds, and methods of using these compounds for treating HIV infection in a subject.BACKGROUND

[0003] Human immunodeficiency virus (HIV), particularly the strains known as HIV type-1 (HIV-1) virus and type-2 (HIV-2) virus, is the etiological agent of the complex disease that includes progressive destruction of the immune sy stem (AIDS) and degeneration of the central and peripheral nervous system. A common feature of HIV retrovirus replication is the insertion by virally-encoded integrase enzyme of +proviral DNA into the host cell genome, a required step in HIV replication in human T-lymphoid and monocytoid cells. Integration is believed to be mediated by HIV integrase in three steps: assembly of a stable nucleoprotein complex with viral DNA sequences; cleavage of two nucleotides from the 3' termini of the linear proviral DNA; and covalent joining of the recessed 3' OH termini of the proviral DNA at a staggered cut made at the host target site. The fourth step in the process, repair synthesis of the resultant gap, may be accomplished by cellular enzymes. Amino acid sequence homology provides evidence that the pol gene of the HIV genome encodes reverse transcriptase, integrase and an HIV protease [Toh, H. et al., EMBO J. 4, 1267 (1985); Power, M.D. et al., Science, 231, 1567 (1986); Pearl, L.H. et al., Nature, 329, 351 (1987)]. Integrase has been shown to be essential for replication of HIV.

[0004] It is known that some antiviral compounds which act as inhibitors of HIV replication are effective agents in the treatment of AIDS and similar diseases, including inhibitors of HIV integrase such as the heterocyclic compounds disclosed in International Publication Nos. WO 2014 / 183532, WO 2018 / 102485, and WO 2022 / 177840. The compounds of this invention have inhibitory activity against HIV integrase, and as such are inhibitors of HIV replication.

[0005] Of interest in the area of HIV therapies and treatments is providing regimens to patients with improved properties, including, for example, potency against prevalent HIV virus mutants, increased half-life, low plasma clearance and / or other properties. While current regimens fortreating HIV have progressed enough that patients no longer have to take multiple pills multiple times a day, they are still required to take a pill every day for the foreseeable span of their life. Thus, it would be beneficial to have HIV therapies that require patients take medication less frequently than once a day, such as once a week, or take a smaller effective dose of the medication on a weekly basis.SUMMARY

[0006] The present disclosure provides fused tricyclic compounds that exhibit activity against HIV integrase, and compositions containing these compounds in isomeric excess. The disclosure also provides methods of making these compounds. Also provided are pharmaceutical compositions comprising any of these compounds, including pharmaceutical compositions adapted for once-weekly administration. Further provided are methods of inhibiting HIV integrase using these compounds. Further provided are methods for treating HIV infection in a subject using these compounds, such as methods for administering these compounds to a subject at a frequency of once-weekly. Also provided are methods for reducing the risk for development of antiviral treatment resistance due to an HIV integrase mutation in a subject infected with HIV, by administering these compounds.

[0007] In some aspects, the present disclosure provides hydroxyl-substituted fused tricyclic compounds. In one aspect, the present disclosure provides compound 1, which has the following formula (1):or a pharmaceutically acceptable salt thereof. In some aspects, the present disclosure provides compositions comprising compound 1. In some aspects, the present disclosure provides compositions comprising a pharmaceutically acceptable salt of compound 1.

[0008] In some aspects, the present disclosure provides compositions comprising compound 1 in isomeric excess. In some embodiments, compositions are provided in which compound 1 is present in an isomeric excess of at least 65%, at least 75%, at least 80%, at least 85%, at least 90%. at least 95%. at least 97.5%, at least 98%, at least 99%, or at least 99.5%. In some aspects, the present disclosure provides compositions comprising compound 1 in enantiomeric excess. In some embodiments, provided are compositions in which compound 1 is present in anenantiomeric excess of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 98%, or at least 99%, or at least 99.5%. In particular embodiments, the disclosed compositions comprise compound 1 and are substantially free of other isomers of compound 1.

[0009] The present invention is directed to compound 1, as well as pharmaceutically acceptable salts, solvates, prodrugs, hydrates, and co-crystals thereof. Compound 1 and pharmaceutically acceptable salts, solvates, hydrates, co-crystals and prodrugs may be useful, for example, for inhibiting HIV viral replication or replicon activity, and / or for treating HIV infection in a subject. Without being bound by any specific theory, it is understood that the compound inhibit HIV viral replication by inhibiting HIV integrase activity.

[0010] Accordingly, the present disclosure provides methods for treating or preventing HIV infection, in a subject in need thereof, by administering to the subject an effective amount of the compound. In one aspect, the disclosure provides methods for the treatment of infection by HIV, or for the treatment or delay in the onset or progression of AIDS, in a subject in need thereof, that comprises administering to the subject an effective amount of compound 1, or a pharmaceutically acceptable salt thereof. Further provided are methods for the inhibition of HIV integrase activity' in a subject in need thereof that comprise administering to the subject an effective amount of compound 1. or a pharmaceutically acceptable salt thereof. In various aspects, the subject is human.

[0011] The present disclosure provides that compound 1 has a long-acting inhibitory activity against HIV integrase. Compound 1 has been administered in vivo to mammalian subjects at frequencies of once-weekly and less than once-weekly. Its pharmacokinetic properties support administration to subjects once-weekly. As such, in some aspects, the disclosed methods involve administration of the compound or salt thereof at a frequency of once-weekly. Accordingly, provided herein are methods for treating infection by HIV, or for treating or delaying the onset or progression of AIDS, in a subject, by administering to the subject once weekly an effective amount the compound 1. or a pharmaceutically acceptable salt thereof.

[0012] Further provided are pharmaceutical compositions comprising compound 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In some aspects, these pharmaceutical compositions are adapted for oral administration to a subject. In particular aspects, these pharmaceutical compositions are adapted for once-weekly oral administration. In some aspects, methods of administering these pharmaceutical compositions to a subject (e.g., a human subject) are provided. Oral administration of compound 1 has thepotential to offer a safe, efficacious, and well-tolerated once-weekly regimen for the treatment of HIV infection and AIDS.

[0013] In some embodiments, provided are pharmaceutical compositions comprising compound 1 as a crystalline solid.

[0014] In some aspects, the disclosed pharmaceutical compositions are present in an oral dosage form, such as a tablet, capsule, or suspension. In one aspect, the composition is present in a tablet or capsule. In some embodiments, the present disclosure provides oral dosage forms comprising an effective amount of compound 1, and a pharmaceutically acceptable carrier. In some aspects, provided are tablets comprising compound 1 and a pharmaceutically acceptable carrier that are adapted for once-weekly oral administration.

[0015] The present disclosure is based, at least in part, on the discovery that compound 1 is a highly active integrase strand transfer inhibitor (INSTI) compound that exhibits an improved antiviral resistance mutation profile relative to existing INSTI compounds. In particular, compound 1 exhibits a minimized potency shift (or "mutant shift’7) against resistance-associated HIV integrase variants, such as the clinically prevalent G140S and Q148H double mutant variant.

[0016] Compound 1 exhibits an improved mutant shift relative to integrase inhibitors in the art. As provided in this disclosure, compound 1 exhibits a 14-fold improvement in potency shift against the GI40S and Q148H double mutant relative to a "racemic" mixture disclosed in International Publication No. WO 2014 / 183532, published November 20, 2014, which is incorporated by reference herein. Compound 1 has a G140S / Q148H mutant shift of 2.9X, which is a small, non-appreciable shift. Accordingly, the present disclosure provides methods of reducing the risk for development of antiviral treatment resistance due to an HIV integrase mutation in a subject infected with HIV, comprising administering an effective amount of compound 1, or a pharmaceutically acceptable salt thereof. In some aspects, the subject is human. Further provided are methods of reducing the risk for development of antiviral treatment resistance due to an HIV integrase mutation by administering any of the disclosed pharmaceutical compositions.

[0017] In another aspect, the present disclosure provides methods of making compound 1, or a pharmaceutically acceptable salt thereof.

[0018] In another aspect, the present disclosure provides uses of compound 1, or a pharmaceutically acceptable salt thereof, including uses of compound 1 in therapy. In some aspects, compound 1 is used in the preparation of a medicament for the inhibition of HIV integrase, for the treatment of infection by HIV, or for the treatment or delay in the onset orprogression of AIDS in a subject (e.g., a human subject) in need thereof. In some embodiments, the disclosed uses comprise once-weekly administration.

[0019] The summary of the technology described above is non-limiting and other features and advantages of the technology will be apparent from the following detailed description, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 depicts the plasma concentration vs. time plots for compound 1 in male Wistar Hannover rats following either an intravenous (“IV”) or an oral (per os, “P. O.”) single administration of compound 1.

[0021] FIG. 2 depicts the plasma concentration vs. time plots for compound 1 in male beagle dogs following either IV or P. O. single dose of compound 1.

[0022] FIG 3 depicts the plasma concentration vs. time plots for compound 1 in male rhesus macaques following either IV or P. O. single dose of compound 1.DETAILED DESCRIPTION

[0023] The present disclosure is directed to an HIV integrase inhibitor compound for the treatment of HIV infection. Provided herein is compound 1, which is (3o / ?.55)-V-t2.4-difluorobenzyl)-2-ethyl-5,8-dihydroxy-l,7-dioxo-2,3,3a,4,5,7-hexahydro-lH-pyrazino[2,l,6-cd]indolizine-6-carboxamide. Compound 1 has a molecular w eight of 419.38. The disclosure further provides methods of making this compound. Further provided herein are pharmaceutical compositions comprising the compound, and methods of administering the compound to a subject in need thereof to inhibit HIV integrase, treat HIV infection, and / or treat AIDS or delay the onset or progression of AIDS.

[0024] Compound 1 exhibits high potency against integrase that is not appreciably shifted in the presence of the GI40S / QI48H double mutant variant. A technical advantage of this compound is maintenance of potency against clinically relevant resistance-associated HIV integrase mutants, including double mutant variants. In contrast, many existing integrase inhibitor compounds fail to maintain potency against resistance-associated mutations in integrase, such as double mutant variants, and instead exhibit a pronounced mutant shift.

[0025] The residue QI 48 in the HIV integrase protein is known to be one of the dominant pathways to INSTI resistance. G140S is a common compensatory mutation that typically occurs after the appearance of a Q148H mutation in the protein. (See Li et al. Mechanisms of HIV-1 integrase resistance to dolutegravir and potent inhibition of drug-resistant variants. Set. Adv. 9,5953 (2023).) While G140S alone has minimal impact on the potency of most INSTls,G140S / Q148H integrase variants can confer greater than 100-fold changes in potency (i.e., potency shifts) in response to raltegravir administration or elvitegravir administration, and a roughly 10-fold potency shift in response to cabotegravir administration. (See Margot NA, et al. Antiviral activity of HIV-1 integrase strand-transfer inhibitors against mutants with integrase resistance-associated mutations and their frequency in treatment-naive individuals. J. Med. Virol.2019; 91:2188-2194; YoshinagaT, etal. Antiviral characteristics of GSK1265744, an HIV integrase inhibitor dosed orally or by long-acting injection. Antimicrob Agents Chemother. 2015; 59(l):397-406.) Additionally, a recent study revealed that the G140S / Q148H mutations were prevalent among 16% of INSTI-resistance clinical samples. (See Gil H, et al. Spanish Group for the Study of Antiretroviral Drug Resistance. Factors associated with HIV-1 resistance to integrase strand transfer inhibitors in Spain: Implications for dolutegravir-containing regimens. Front Microbiol. 2022 Dec 12; 1 3:1051096.) Thus, GMOS and Q148H are highly clinically relevant integrase resistance variants. There is a need in the art for active INSTI compounds that exhibit an improved resistance mutation profile, e.g., against variants GMOS and Q148H, in subjects to wftich the compound(s) is administered.

[0026] Compound 1 exhibits an improved resistant mutation profile relative to existing integrase inhibitors. As provided in Example 4, this compound exhibits a 14-fold improvement in potency shift in vitro against the G140S / Q148H double mutant relative to a racemate disclosed in International Publication No. WO 2014 / 183532. This publication provides 38 illustrated compound structures. The present invention involves the discovery and determination that one particular structure represents an isomeric mixture containing four stereoisomers (“the isomeric mixture’'). The inventors have designed syntheses to generate and isolate these four stereoisomers (see Example 2). Compound 1 is one of those stereoisomers. The other three are Compound 2, Compound 3, and Compound 4, whose chemical structures are illustrated below. These four compounds can be isolated from each other through, e.g., chiral separation or by boiling point.Compound 3 Compound 4

[0027] The isomeric mixture exhibits activity against integrase. However, this isomeric mixture was found to have a G140S / Q148H mutant shift of 40.6X, as determined by the VIKING assay (see Example 4). That is, in order to achieve equivalent potency against the double mutant relative to a wild-type integrase, a 40.6-fold greater dose is required.

[0028] Generally, it is unlikely that an anti-HIV compound that e.xhi bi ts a potency shift in a clinically prevalent double mutant (e.g., two resistance mutations in integrase) of 13X or more would be developed as a drug product. See, e.g., Margot NA, et al. J. Med. Virol. (2019); Smith et al. ACS Infect Dis. (2021) 7. 1469-1482; and Jiang et al. Comms. Chem. (2023) 6:83, citing Huang, et al. J. Med. Chem. 62, 2083-2098 (2019). Such a scenario would require a significantly high dose to achieve therapeutic efficacy in subjects having HIV that present clinically prevalent resistance mutations.

[0029] Compound 1 surprisingly has a G140S / Q148H mutant potency shift of only about 3X (2.9X in 10% normal human serum, VIKING), which is a shift sufficiently small to be acceptable for development as an HIV treatment at minimal doses. Compound 1 exhibits an excellent potency (IC50) against wild-type integrase of 42.72 nM (in 10% normal human serum, see Example 4, Table 1). In order to achieve equivalent potency against the double mutant, it is anticipated that only a2.9-greater dose of compound 1 is required. Compound l's double mutant shift of 2.9X represents a 14-fold reduction from its isomeric mixture. Likewise, shifts of 1 (no measurable shift) and even less than 1 were observed in vitro in response to single mutant viruses, such as a Q148 mutation individually. As such, compound 1 maintains high potency against HIV integrase, even in the presence of integrase resistance mutations.

[0030] In addition, compound 1 exhibits an excellent potency in the absence of normal human serum against wild-type integrase of 1.4 nM, as measured by VIKING.

[0031] In addition to its virological properties, the disclosed compound 1 and its pharmaceutically acceptable salts exhibit favorable PK properties that are improved relative to existing integrase inhibitor compounds. In exemplary embodiments, compound 1 exhibits long plasma half-life in (non-clinical) mammalian subjects in vivo, including in non -human primate (NHP) subjects. In addition, compound 1 exhibits long plasma mean residence time (MRT) in mammalian subjects. In exemplary embodiments, compound 1 exhibits improved bioavailability in mammalian subjects, such as NHP subjects.

[0032] In exemplary embodiments, compound 1 exhibits therapeutically effective plasma clearance in mammalian subjects in vivo, including in NHP subjects. In some embodiments, compound 1 may exhibit an improved clearance in HIV-positive subjects and / or smoking human subjects (“smokers”) relative to existing integrase inhibitor compounds, such as existing orally administered compounds.In some aspects, methods of making compound 1, or a pharmaceutically acceptable salt thereof, are provided herein. In some embodiments, the disclosed methods of making involve one or more iodinated intermediates. In some embodiments, these methods involve installation of a hydroxygroup after an iodination step (see intermediates Int-G and Int-H of Example 1). In some embodiments, the iodination step is performed by adding N-iodosuccinimide (NIS) to the reaction mixture (e.g. containing Int-F). As such, provided herein are methods of making compound 1, or a pharmaceutically acceptable salt thereof, comprising a step of iodinating 8-(benzyloxy)-2-ethyl-3,3a,4,5-tetrahydro-l / 7-pyrazino[2, 1,6-c< ]indolizine-l,7(277)-dione (Int-F) to generate 8-(benzyloxy)-2-ethyl-6-iodo-3,3a,4,5-tetrahydro-l / / -pyrazino[2, l,6-c< / ]indohzine-l,7(2Z / )-dione (Int-G). In particular embodiments, the method further comprises a step of installing a hydroxy group onto the 8-(benzyloxy)-2-ethyl-6-iodo-3,3a,4,5-tetrahydro-177-pyrazino|2. l.6-c<7|indolizine- l.7(2 / 7)-dione product of this reaction. This step generates Int-H.

[0033] In some embodiments, the disclosed methods may be used to make amounts of about 100, 150, 200, 215, 230, 250, 300, or more than 300 grams of compound 1.

[0034] In another aspect, methods of making stereoisomers of compound 1 are also provided. In some aspects, methods of making compound 2 are provided. In some aspects, methods of making compound 3 are provided. In some aspects, methods of making compound 4 are provided.

[0035] The disclosed methods are useful for generating compositions comprising compound 1 in isomeric excess and / or enantiomeric excess. Such compositions are useful in that they are substantially free of other compounds, such as isomers of compound 1.

[0036] Synthesis of the racemate was reproduced in accordance with the procedure described in International Publication No. WO 2014 / 183532. This procedure generated a mixture containing compounds 1, 2, 3 and 4. As such, this mixture contains compound 1 in an amount of less than 40% among the four isomers. Since compounds 1 and 4 are enantiomers of one another, this mixture generated equal parts (1:1 ratio) of compound 1 and its enantiomer. As such, this procedure did not contain compound 1 in any enantiomeric excess.

[0037] Thus, the above-referenced and reproduced procedure failed to produce compound 1 in any isomeric or enantiomeric excess. This procedure generated less than 40% of compound 1 among the four isomers. It generated no more than 50% of compound 1 as between it and its enantiomer compound 4.

[0038] In various embodiments, these compositions comprise compound 1 in an isomeric excess (or diastereomeric excess) of at least 50%, at least 55%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 98%, or at least 99%, or at least 99.5% (e.g., w / w). These compositions may contain compound 1 in an isomeric excess of at least 65%. In some embodiments, these compositions contain compound 1 in an at least 75%, 80%, 85%, or 90% isomeric excess. In some aspects, the disclosed compositions comprise compound 1 is an isomeric excess of 98%. In some aspects, the disclosed compositions comprise compound 1 in an isomeric excess of approximately 100%.

[0039] In various embodiments, these compositions comprise compound 1 in an enantiomeric excess of at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 98%, or at least 99%, or at least 99.5% (e.g., w / w). These compositions may contain compound 1 in an enantiomeric excess of at least 75%. In some embodiments, these compositions contain compound 1 in an at least 80%, 85%, or 90% enantiomeric excess. In some aspects, the disclosed compositions comprise compound 1 is an enantiomeric excess of 98%. In some aspects, the disclosed compositions comprise compound 1 is an enantiomeric excess of approximately 100%.

[0040] In some aspects, the disclosed compositions are substantially free of isomers of compound 1. In exemplary embodiments, compositions comprising compound 1 are provided that are substantially free of any of compound 2, compound 3 or compound 4. In some aspects, the disclosed compositions consist essentially of compound 1.

[0041] In some aspects, the present disclosure also provides tricyclic heterocycle compounds for use (i) in, (ii) as a medicament for, or (iii) in the preparation of a medicament for: (a) therapy; (b) inhibiting HIV replication or (c) treating HIV infection and / or reducing the likelihood or severity of symptoms of HIV infection. In these uses, the compound of the present invention may be employed in combination with one or more second therapeutic agents selected from HIV antiviral agents, anti -infective agents, and immunomodulators.

[0042] The present disclosure further provides oral dosage forms for use in any of the methods described herein, such as once-weekly administration to a subject for treatment of HIV and / or reducing the likelihood or severity of symptoms of HIV infection. In some aspects, provided herein is a tablet for use in any of the methods described herein. The present disclosure further provides use of a tablet, for the preparation of a medicament for use in any of the methods described herein. The present disclosure further provides a capsule, for use in any of the methods described herein or for the preparation of a medicament for use in any of the methods described herein. The present disclosure further provides an orally administrable suspension, for use in any of the methods described herein.

[0043] The tricyclic heterocycle compounds disclosed herein, including compound 1, may be useful in human medicine for treating or preventing HIV infection in a subject. In exemplary embodiments, the tricyclic heterocycle compounds inhibit HIV-1 viral replication. Accordingly, the tricyclic heterocycle compounds may be useful for treating HIV infections and AIDS. In accordance with the invention, the tricyclic heterocycle compounds may be administered to a subject in need of treatment of HIV infection.

[0044] Accordingly, the disclosure provides methods for treating HIV infection in a subject comprising administering to the subject an effective amount of at least one tricyclic heterocycle compound or a pharmaceutically acceptable salt thereof. In a specific embodiment, the present invention provides methods for treating AIDS in a subject comprising administering to the subject an effective amount of at least one tricyclic heterocycle compound or a pharmaceutically acceptable salt thereof.Definitions

[0045] Listed below are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

[0047] As used herein, the articles “a” and '“an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0048] As used herein, the term “about” in quantitative terms refers to plus or minus 10% of the value it modifies (rounded up to the nearest whole number if the value is not sub-dividable, such as a number of molecules or nucleotides).

[0049] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 50 mg to 500 mg” is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0050] As used herein, the term “comprising” may include the embodiments “consisting of’ and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “may,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as “consisting of and “consisting essentially of the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.

[0051] As used herein, the term “composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

[0052] One or more compounds of the invention may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and it is intended that the invention embrace both solvated and unsolvated forms. “Solvate” refers to a physical association (e.g., complex) of a compound of this invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation, forexample when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. This term encompasses both solution-phase and isolatable solvates. Nonlimiting examples of solvates include ethanolates, methanolates, and the like. A "hydrate" is a solvate in which the solvent molecule is water. That is, a ‘‘hydrate’' refers to a complex formed by the combining of a disclosed compound and water. In some embodiments, the compound of the invention is provided in hydrate form, such as a monohydrate form (a complex of molecule of water and the compound in a 1: 1 ratio).

[0053] One or more compounds of the invention may optionally be converted to a solvate. Preparation of solvates is generally known. Thus, for example, M. Caira et al. J. Pharmaceutical Sci., 93(3), 601-611 (2004) describe the preparation of the solvates of the antifungal fluconazole in ethyl acetate as well as from water. Similar preparations of solvates, hemisolvate, hydrates and the like are described by E. C. van Tonder et al. AAPS PharmSciTechours., 5(1), Art. 12 (2004); and A. L. Bingham et al. Chem. Commun., 603-604 (2001). A typical, non-limiting, process involves dissolving the inventive compound in desired amounts of the desired solvent (organic or water or mixtures thereof) at a higher than room temperature, and cooling the solution at a rate sufficient to form crystals which are then isolated by standard methods. Analytical techniques such as, for example IR spectroscopy, show the presence of the solvent (or water) in the crystals as a solvate (or hydrate).

[0054] As used herein, the term "co-crystal" refers to a crystalline material formed by combining a compound disclosed herein and one or more co-crystal formers (i.e., a molecule, ion, or atom). In certain instances, co-crystals may have improved properties as compared to the parent form (z.e., the free molecule, zwitterion, etc.) or a salt of the parent compound. Improved properties can be increased solubility, increased dissolution, increased bioavailability. increased dose response, decreased hygroscopicity, a crystalline form of a normally amorphous compound, a crystalline form of a difficult-to-salt compound, decreased form diversity, more desired morphology, and the like. Methods for making and characterizing co-crystals are known to those of skill in the art.

[0055] The compound of the disclosure can form salts which are also within the scope of this invention. Reference to a compound herein is understood to include reference to salts thereof, unless otherwise indicated. The term “salt(s),” as employed herein, denotes acidic salts formed with inorganic and / or organic acids, as well as basic salts formed with inorganic and / or organic bases. In addition, when a tricyclic heterocycle compound contains both a basic moiety, such as, but not limited to a pyridine or imidazole, and an acidic moiety, such as, but not limited to a carboxylic acid, zwitterions ("inner salts") may be formed and are included within the term"salt(s)" as used herein. In one embodiment, the salt is a pharmaceutically acceptable (i.e., nontoxic, physiologically acceptable) salt. In another embodiment, the salt is other than a pharmaceutically acceptable salt. Salts of the Compound 1 may be formed, for example, by reacting a tricyclic heterocycle compound with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.

[0056] Exemplary salts include acetates, ascorbates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, fumarates, hydrochlorides, hydrobromides, hydroiodides, lactates, maleates, methanesulfonates, naphthalenesulfonates, nitrates, oxalates, phosphates, propionates, salicylates, succinates, sulfates, tartarates, thiocyanates, toluenesulfonates (also known as tosylates) and the like, which are all acid salts. All such acid salts and base salts are intended to be pharmaceutically acceptable salts within the scope of the invention and all acid and base salts are considered equivalent to the free forms of the corresponding compounds for purposes of the invention.

[0057] Diastereomeric, or isomeric, mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods well-known to those skilled in the art, such as, for example, by chromatography and / or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a isomeric mixture by reaction with an appropriate optically active compound (e.g., chiral auxiliary such as a chiral alcohol or Mosher’s acid chloride), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers.Sterochemically pure compounds may also be prepared by using chiral starting materials or by employing salt resolution techniques. Enantiomers can also be directly separated using chiral chromatographic techniques.

[0058] A “stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The present disclosure contemplates various stereoisomers and mixtures thereof and includes “enantiomers”, which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0059] “Enantiomers” are a pair of stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is known as a “racemic” mixture. The term “enantiomeric excess” (“ee”) refers to an isomeric mixture in which in a majority (more than 50%) of the mixture constitutes a particular enantiomer (e.g., as a weight-over-weight, w / w).Enantiomeric excess may be measured using any method known in the art, e.g., chiral liquid chromatography with mass spectrometry (chiral LC-MS) for w / w measurements.

[0060] As used herein, the terms “isomeric excess” and “diastereomeric excess” (“de”) refer to an isomeric mixture in which in a majority (more than 50%) of the mixture constitutes a particular stereospecific compound (e.g., w / w). Isomeric excess may be measured using any method known in the art, e.g., chiral LC-MS for w / w measurements.

[0061] It is also possible that the compound may exist in different tautomeric forms, and all such forms are embraced within the scope of the invention. For example, all keto-enol tautomeric forms of the disclosed compounds are included in the invention. Thus, provided herein are tautomers of compound 1, and pharmaceutically acceptable salts thereof.

[0062] Individual stereoisomers of the compound of the disclosure may, for example, be substantially free of other isomers. For instance, compositions comprising Compound 1 are provided that are substantially free of other isomers of Compound 1. As used herein, a composition that is “substantially free” of a particular compound(s) refers to a composition having 3% or less, 2% or less, 1% or less, e.g., 0.5% or less, of that compound(s) as measured by w / w or volume / volume (v / v). Preferably, chiral LC-MS is used to measure the amount (%) in w / w of a particular compound in a mixture.

[0063] In Compound 1, the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. For example, different isotopic forms of hydrogen (H) include protium (¹H) and deuterium (2H). Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterization of biological samples. Isotopically -enriched Compound 1 can be prepared without undue experimentation by conventional techniques well known to those skilled in the art or by processes analogous to those described in the Schemes and Examples herein using appropriate isotopically-enriched reagents and / or intermediates. In one embodiment, Compound 1 has one or more of its hydrogen atoms replaced with deuterium.

[0064] Polymorphic forms of the disclosed hydroxyl tricyclic heterocycle compounds, and of the salts, solvates, hydrates, esters, co-crystals and prodrugs of the disclosed compounds, are intended to be included in the present invention.

[0065] The compound can be administered in the form of a pharmaceutically acceptable salt. The term “pharmaceutically acceptable salt” refers to a salt which possesses the effectiveness ofthe parent compound and which is not biologically or otherwise undesirable (e.g., is neither toxic nor otherwise deleterious to the recipient thereof). Suitable salts include acid addition salts which may, for example, be formed by mixing a solution of the compound of the present invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, acetic acid, or benzoic acid. When compounds employed in the present invention carry an acidic moiety (e g., -COOH or a phenolic group), suitable pharmaceutically acceptable salts thereof can include alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., calcium or magnesium salts), and salts formed with suitable organic ligands such as quaternary' ammonium salts. Also, in the case of an acid (-COOH) or alcohol group being present, pharmaceutically acceptable esters can be employed to modify the solubility or hydrolysis characteristics of the compound.

[0066] The term "administration" and variants thereof (e.g., "administering" a compound) in reference to a compound of Formula 1 mean providing the compound to the individual in need of treatment or prophylaxis. When a compound is provided in combination with one or more other active agents (e.g., antiviral agents useful for treating or prophylaxis of HIV infection or AIDS), "administration" and its variants are each understood to include provision of the compound and other agents at the same time or at different times. When the agents of a combination are administered at the same time, they can be administered together in a single composition or they can be administered separately.

[0067] As used herein, the term "pharmaceutical composition" is intended to encompass a product comprising the specified ingredients, as well as any product which results from combining the specified ingredients. Ingredients suitable for inclusion in a pharmaceutical composition are pharmaceutically acceptable ingredients, which means the ingredients must be compatible with each other and not deleterious to the recipient thereof.

[0068] As used herein, the term “pharmaceutically acceptable carrier” is meant to refer to any adjuvant, vehicle, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0069] As used herein, the term “subject” refers to an animal, preferably a mammal, that has been the object of treatment by or administration of a compound. In exemplary embodiments, the subject is a mammal. In some embodiments, the subject is a laboratory animal. In some embodiments, the subject is a domesticated animal. In some embodiments, the subject is a primate. The subject may a rodent, such as a rat. In some embodiments, the subject is human. Insome embodiments, the subject is a dog (e.g., a beagle dog). In some embodiments, the subject is a non-human primate (NHP), such as a rhesus macaque.

[0070] As used herein, the term “resistance mutation” refers to a mutation in the DNA sequence encoding an HIV protein that is associated with antiviral resistance and arises due to the inhibitory pressure of an agent, such as a treatment-emergent resistance-associated mutation. “Resistance-associated mutation” refers to an HIV protein mutation that confers resistance to the agent when treated with escalating doses of the agent. In some aspects, this term refers to a resistance-associated mutation in the HIV integrase protein. This term encompasses mutations that arise in subjects to which an anti -HIV compound has been administered, whether or not prevalent in one or more clinical populations. This term encompasses mutations that arise in treatment-naive subjects or treatment-experienced subjects. This term further encompasses multidrug resistance-associated mutations.

[0071] The term "effective amount" as used herein means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, medical doctor or other clinician. In one embodiment, the effective amount is a "therapeutically effective amount" for the alleviation of the symptoms of the disease or condition being treated. The term also includes herein the amount of active compound sufficient to inhibit HIV integrase (wild type and / or mutant strains thereof) and thereby elicit the response being sought. When the active compound (i.e., active ingredient) is administered as the salt, references to the amount of active ingredient are to the free form (i.e., the non-salt form) of the compound.

[0072] As used herein, “mean residence time (MRT)” refers to the average total time that a given dose of an agent is present in the body of a subject and is calculated as the area under the first moment curve (AUMC) / area under the cune (AUC). The AUC variable may be expressed as AUCo-inf, the area under the concentration-time curve from time = 0 to the last quantifiable concentration and extrapolated to infinity. Areas under the (plasma concentration) curve may be calculated by any method known in the art, such as analytical methods such as LC-MS / MS, used to quantify plasma concentration over a given time course, and may be analyzed by, e.g., noncompartmental methods, utilizing software tools such as Watson LIMS®.

[0073] As used herein, the terms “mean plasma half-life (ti / 2)” and “terminal half-life (ti / 2)” refer to the average time it takes for one-half of an administered agent to be reduced from its original value. It is used to estimate the time it takes to eliminate the agent from the plasma of the subject through biological processes, e.g., metabolism, measured after the initial dose.

[0074] As used herein, the term “volume of distribution7’ refers to the distribution and degree of retention of an agent throughout the various compartments of the body of a subject. This term may be expressed as the “volume of distribution at steady state (Vdss)”, which is the estimated volume of the body into which the agent has distributed at steady state. A large Vdss suggests the agent has distributed more broadly throughout the body and may be associated with longer plasma half-life.

[0075] As used herein, the term “clearance (CL)” refers to the rate at which an administered agent is cleared from the body, e.g., through excretion, elimination, or the plasma (CLP).Pharmaceutical Compositions

[0076] In some aspects pharmaceutical compositions comprising compound 1, or a pharmaceutically acceptable salt thereof, are provided herein.

[0077] In some embodiments, the pharmaceutical compositions are adapted for oral administration. As such, oral dosage forms containing the disclosed pharmaceutical compositions are contemplated. In some embodiments, the compositions are provided as a solid oral dosage form, such as a tablet, capsule, or mini-tablet. In exemplary embodiments, the compositions are provided as one or more tablets. In some embodiments, the compositions are provided as one or more capsules.

[0078] In some aspects, tablets comprising any of the disclosed compounds or pharmaceutical compositions, and a pharmaceutically acceptable carrier, are provided. In some aspects, capsules comprising any of the disclosed compounds or pharmaceutical compositions, and a pharmaceutically acceptable carrier, are provided. In some embodiments, the tablet or capsule comprises a solvate of compound 1, such as a hydrate.

[0079] In some embodiments, the tablet comprises one or more pharmaceutically acceptable excipients selected from mannitol, microcrystalline cellulose, croscarmellose sodium, and magnesium stearate, or any combination thereof. In some embodiments, the tablet comprises mannitol, microcrystalline cellulose, croscarmellose sodium, and magnesium stearate.

[0080] The tablets disclosed herein may be uncoated or coated (in which case they include an outer film coat). Although uncoated tablets may be used, it is more usual to provide a coated tablet, in which case a conventional non-enteric coating may be used. Film coatings are known in the art and can be composed of hydrophilic polymer materials, but are not limited to, polysaccharide materials, such as hydroxypropylmethyl cellulose (HPMC), methylcellulose, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), poly(vinylalcohol-co-ethylene glycol) and other water-soluble polymers.

[0081] In some embodiments, any of the disclosed dosage forms (e.g., one or multiple tablets or capsules) comprises about 100 mg to about 1000 mg of compound 1, or a pharmaceutically acceptable salt, for example, about 100 to 250 mg, about 250 to 500 mg, about 200 to 600 mg, about 250 to 750 mg, about 500 to 750 mg, about 750 to 1000 mg, or about 500 to 1000 mg.

[0082] Two or more tablets or capsules may be provided to a subject (e.g., a human subject) in a single administration. For instance, a single tablet may be administered. In other embodiments, more than one tablet is administered. In some embodiments, two tablets are administered. In some embodiments, three tablets are administered.

[0083] Each tablet or capsule may comprise between about 100 to 200 mg, about 100 to 250 mg, about 100 to 300 mg, about 100 to 400 mg, about 100 to 500 mg, about 100 mg to 600 mg, about 100 mg to 650 mg, about 250 to 500 mg, about 200 to 600 mg, or about 300 to 600 mg, about 400 to 600 mg, about 400 to 650 mg, or about 500 to 600 mg, of compound 1, or a pharmaceutically acceptable salt thereof. In some embodiments, the tablet or capsule comprises 600 mg of compound 1, or a pharmaceutically acceptable salt thereof.

[0084] In some embodiments of the disclosed solid oral dosage forms (e g., tablets or capsules), the pharmaceutical composition is present in the dosage form as a micro-suspension. In particular embodiments, the provided solid oral dosage form comprises methylcellulose and / or sodium lauryl sulfate (SLS). In some embodiments, any of these dosage forms comprises Tween, e.g., Tween80. In some embodiments, any of these dosage forms comprises Tween, methylcellulose, and SLS. For example, any of these dosage forms may comprise methylcellulose in a 0.5% (v / v) amount, SLS, and Tween.

[0085] In some embodiments of the disclosed pharmaceutical compositions, the compositions contain compound 1 as a crystalline solid. In some embodiments, compound 1 is present as an anhydrous crystalline solid.

[0086] In other aspects, the disclosed pharmaceutical compositions are present in an oral liquid suspension, such as an aqueous suspension. In particular embodiments, the provided suspension comprises methylcellulose and / or SLS. The provided suspension may comprise methylcellulose in a 0.5% (v / v) amount. The provided suspension may comprise methylcellulose and further comprise SLS, e.g., in an amount of 0.1% or 0.25% (v / v). In some embodiments, the provided suspension comprises Tween, e.g., Tween80. In another embodiment, the composition is provided in an oral suspension comprising polyethylene glycol, such as PEG400. The disclosed PEG400 suspensions may be provided in water or DMSO solvent.

[0087] Liquid preparations suitable for oral administration (e.g., suspensions, syrups, elixirs and the like) can be prepared according to techniques known in the art and can employ any of theusual media such as water, glycols, oils, alcohols and the like. Solid preparations suitable for oral administration (e.g., powders, pills, capsules and tablets) can be prepared according to techniques known in the art and can employ such solid excipients as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like.

[0088] Any of the disclosed pharmaceutical compositions may, following a single administration, provide a mammalian subject (e g., a human subject) a mean plasma half-life of at least 90 hours, at least 100 hours, at least 125 hours, at least 150 hours, at least 165 hours, at least 175 hours, at least 200 hours, or at least 225 hours. In some embodiments, any of these compositions provides a mean half-life of 90 hours or greater. These compositions may provide a mean half-life of 100 hours or greater. These compositions may provide a mean half-life of 125 hours, 150 hours, 170 hours, 180 hours, 200 hours, or greater. In exemplary embodiments, these compositions provide a mean half-life of about 100 hours (e.g., 103 hours), 185 hours, or 190 hours (e.g.. 193 hours) in a mammalian (e g., an NHP or human) subject. In some embodiments, the disclosed compositions may exhibit a therapeutically effective (e.g., a relatively long halflife, despite any observed decrease in the) plasma half-life in smokers.

[0089] The plasma half-life and mean residence time (MRT) in mammalian subjects following administration of compound 1 correlate well. In some embodiments, any of the disclosed pharmaceutical compositions may, following a single administration, provide in a mammalian subject (e.g., a human subject) an MRT of about 100 hours, 125 hours, 140 hours, 150 hours, 175 hours, 200 hours, 215 hours, 225 hours, 240 hours, or greater. In some embodiments, an MRT of 100-125, 100-140, 100-150, 100-175, 175-225, 200-225, 200-250, 100-200, or 100-250 hours is provided.

[0090] In some aspects, any of the disclosed pharmaceutical compositions may exhibit an average absolute bioavailability (F) in a mammalian subject (e.g., a human subject) of about 15%, 20%, 25%, 30%, 35%, 40%, or greater than 40%. In some embodiments, an F of 15-25%, 25-35%, 20-30%, 20-40%, or 20-50% is provided for any of the disclosed pharmaceutical compositions in mammalian subjects.

[0091] In some aspects, any of the disclosed pharmaceutical compositions may exhibit a plasma clearance (CLP) in a mammalian subject (e.g., a human subject) of about 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.010, 0.012, 0.015, or 0.020 ml / min / kg body weight of subject. In some embodiments, a CLPof about 0.010 ml / min / kg is provided. In some embodiments, the disclosed compositions may exhibit a therapeutically effective plasma clearance in smokers (e.g., a relatively low clearance despite any observed increase in clearance).Methods of Treatment

[0092] In the methods of the present invention (i.e., inhibiting HIV integrase, treating HIV infection or treating or delaying the onset or progression of AIDS), Compound 1, optionally in the form of a salt, can be administered by any means that produces contact of the active agent with the agent’s site of action. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents. They can be administered alone, but typically are administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

[0093] Accordingly, in some aspects are provided methods for inhibiting HIV integrase by administering compound 1, or a pharmaceutically acceptable salt thereof, to a subject. In various aspects, an amount of compound 1 or a pharmaceutically acceptable salt thereof effective to treat HIV infection or AIDS is administered to a subject, such as a subject in need thereof (e.g., a human subject suffering from or living with HIV infection). In exemplary aspects, provided are methods for treating HIV infection in a subject in need thereof by administering compound 1, or a pharmaceutically acceptable salt thereof. In additional aspects, provided are methods for providing treatment of delay in the onset or progression of AIDS in a subject in need thereof, by administering compound 1, or a pharmaceutically acceptable salt thereof. In additional aspects, provided are methods for providing treatment of delay in the onset or progression of AIDS-related complexes (ARC) in a subject in need thereof, by administering compound 1, or a pharmaceutically acceptable salt thereof.

[0094] In additional aspects, provided herein are methods of reducing the risk for development of antiviral treatment resistance due to an HIV integrase mutation in a human subject infected with HIV by administering compound 1, or a pharmaceutically acceptable salt thereof. In some embodiments, provided are methods of reducing the risk for development of antiviral treatment resistance due to two or more HIV integrase mutations in a human subject infected with HIV. In some embodiments, provided are methods of reducing the risk for development of antiviral treatment resistance due to HIV integrase mutations that arise in treatment-experienced subjects. In particular embodiments, provided are methods of reducing the risk for development of antiviral treatment resistance due to HIV integrase mutations that arise in HIV treatment-experienced subjects. In some embodiments, provided are methods of reducing the risk for development of antiviral treatment resistance due to HIV integrase mutations that arise in INSTI treatment-experienced subjects. In some embodiments, provided are methods of reducing the riskfor development of antiviral treatment resistance due to mutations in the G140 and QI 48 positions, such as the prevalent G140S and Q148H substitutions.

[0095] Reduction in the risk for development of antiviral resistance may be measured by any method known in the art, such as in vitro and in vivo measurements of shifts in potency against cells infected by viruses exhibiting one or more resistance-associated mutations. Exemplary in vitro measurements may be made in the absence of serum, such as normal human serum (NHS); or in the presence of NHS, such as 10% NHS or 100% NHS.

[0096] The pharmaceutical compositions may be administered in either single or multiple doses. The pharmaceutical compositions may be administered by various methods including, for example, oral, rectal, buccal, intranasal, parenteral and transdermal routes. In exemplary embodiments, the pharmaceutical compositions may, for example, be administered orally, e.g., in an oral dosage form. The compositions may be administered orally in the form of a unit dosage of a pharmaceutical composition containing an effective amount of the compound and a pharmaceutically acceptable carrier.

[0097] Administration of compound 1, or a pharmaceutically acceptable salt thereof, can be carried out via any of the accepted modes of administration of agents for serving similar utilities. In some embodiments, the administration is oral administration.

[0098] The compound of the disclosure may be administered as a single dose, once-daily or less frequently. In various aspects, the compound may be administered as a single dose, once-weekly. The compound may be administered as multiple or divided doses, once-weekly.

[0099] In some embodiments, the disclosed methods comprise orally administering to the subject compound 1, or a pharmaceutically acceptable salt thereof, or any of the disclosed pharmaceutical compositions, once weekly. In some embodiments of these methods, any of the disclosed pharmaceutical compositions are provided in an oral dosage form. In particular embodiments, these compositions are provided in a tablet or capsule. In some embodiments, the compositions are provided in one or more tablets or capsules once weekly. In exemplary embodiments, the compositions are provided in one, two. or three tablets administered to the subject once weekly. In some embodiments, the compositions are provided in a single tablet (or capsule).

[0100] In some embodiments of the disclosed methods, the pharmaceutical compositions are provided in an oral suspension, such as an aqueous suspension. In particular embodiments, the provided suspension comprises methylcellulose and / or sodium lauryl sulfate (SLS).

[0101] In exemplary aspects of the disclosed methods, the subject is a mammalian subject. In some aspects, the subject is human. In additional aspects, the subject is an NHP. In additional aspects, the subject is a dog. In additional aspects, the subject is a rodent, such as a rat.

[0102] In some aspects of the disclosed methods, administration of an effective amount of compound 1. or a pharmaceutically acceptable salt thereof, to a mammalian subject (e.g., a human subject) provides a mean plasma half-life of at least 90 hours, at least 100 hours, at least 125 hours, at least 150 hours, at least 175 hours, at least 200 hours, or at least 225 hours. In some embodiments, administration provides a mean half-life of 90 hours or greater. In some embodiments, administration provides a mean half-life of 100 hours or greater. Administration of an effective amount of any of the compound may provide a mean half-life of 125 hours, 150 hours, 170 hours, 180 hours, 200 hours, or greater. In exemplary embodiments, administration provides a mean half-life of about 100 hours (e.g., 103 hours), 185 hours, or 190 hours (e.g., 193 hours) in a mammalian subject.

[0103] In some aspects of the disclosed methods, administration of an effective amount of compound 1, or a pharmaceutically acceptable salt thereof, to a mammalian subject (e.g., a human subject) may provide a mean residence time (MRT) of about 100 hours, 125 hours, 140 hours, 150 hours, 175 hours, or greater.

[0104] In some embodiments, any of the disclosed methods of treatment comprises administering to a subject between about 100 mg to about 2250 mg of compound 1, or a pharmaceutically acceptable salt, for example, about 100 to 250 mg, about 250 to 500 mg, about 200 to 600 mg, about 250 to 750 mg, about 500 to 750 mg, about 750 to 900 mg about 750 to 1000 mg, about 500 to 1000 mg. about 750 to 1250 mg. about 800 mg to 1200 mg. about 800 mg to 1600 mg, about 1000 to 1200 mg, about 1000 to 1500 mg, about 500 to 1500 mg, about 1000 to 2000 mg, about 1500 to 2000 mg, about 750 to 1500 mg, about 1000 to 1250 mg, about 1250 to 1750 mg, about 1750 to 2000 mg, about 2000 to 2250 mg, about 2000 to 2500 mg, or about 100 to 2250 mg, of compound 1, or a pharmaceutically acceptable salt thereof.

[0105] In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered orally in a single dose of about 100 mg once weekly. In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered orally in a single dose of about 150 mg once weekly. In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered orally in a single dose of about 200 mg once weekly. In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered orally in a single dose of about 250 mg once weekly. Compound 1, or a pharmaceutically acceptable salt thereof, may be administered orally in a single dose of about 300 mg onceweekly. In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered orally in a single dose of about 350 mg once weekly. Compound 1, or a pharmaceutically acceptable salt thereof, may be administered orally in a single dose of about 400 mg once weekly. In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered orally in a single dose of about 450 mg once weekly. In some embodiments, compound 1, or a pharmaceutically acceptable salt thereof, is administered orally in a single dose of about 500 mg once weekly. Compound 1, or a pharmaceutically acceptable salt thereof, may be administered orally in a single dose of about 600 mg once weekly.

[0106] The frequency of dosage of the compound of the present disclosure, or a pharmaceutically acceptable salt thereof, will be determined by the needs of the individual patient and can be, for example, once per day, once per week, or once per month. Administration of the compound, or a pharmaceutically acceptable salt thereof, continues for as long as necessary to treat the HIV infection, or any other indication described herein. For example, a compound, or a pharmaceutically acceptable salt thereof, can be administered to a human suffering from an HIV infection, for the duration of the human’s life.

[0107] Administration can be intermittent, with a period of several or more days during which a patient receives a daily dose of the compound of the present disclosure, or a pharmaceutically acceptable salt thereof, followed by a period of several or more days during which a patient does not receive a daily dose of the compound or a pharmaceutically acceptable salt thereof. For example, a patient can receive a dose of the compound, or a pharmaceutically acceptable salt thereof, every other day, or three times per week. Again by way of example, a patient can receive a dose of the compound, or a pharmaceutically acceptable salt thereof, each day for a period of from 1 to 14 days, followed by a period of 7 to 21 days during which the patient does not receive a dose of the compound, or a pharmaceutically acceptable salt thereof, followed by a subsequent period (e.g., from 1 to 14 days) during which the patient again receives a daily dose of the compound, or a pharmaceutically acceptable salt thereof.

[0108] Alternating periods of administration of the compound, or a pharmaceutically acceptable salt thereof, followed by non-administration of the compound, or a pharmaceutically acceptable salt thereof, can be repeated as clinically required to treat the patient.

[0109] The disclosed compounds can be administered orally in a dosage range of 0.001 to 1000 mg / kg of mammal body weight in a single dose or in divided doses. One dosage range is 0.01 to 500 mg / kg body weight orally in a single dose or in divided doses. Another dosage range is 0.1 to 100 mg / kg, or 0.5 to 50 mg / kg, body weight orally in single or divided doses. Exemplary dosagesfor larger mammals include 1, 5, 10, 15, 20, 25, 30, 50, and 100 mg / kg body weight. These dosages may be administered once-weekly, or at a different frequency.

[0110] For oral administration, the compositions can be provided in the form of tablets or capsules containing 100 to 2000 milligrams of the active ingredient, particularly 100, 150, 200, 250, 300. 350, 400, 450. 500, 550. 600, 650, 700, 800, 900. 1000, 1250, 1500, 1750, or 2000 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the PK properties of that compound, the age. body weight, general health, sex, diet, mode and time of administration, drug combination, the severity of the particular condition, and the subject undergoing therapy.Combination Therapies

[0111] In some aspects, any of the disclosed methods for treating HIV infection, treating AIDS, delaying the onset or progression of AIDS, and inhibiting HIV integrase may further comprise the administration of one or more additional therapeutic agents that is not compound 1, or a pharmaceutically acceptable salt thereof. In some aspects, the present pharmaceutical compositions can further comprise the administration of one or more additional therapeutic agents that is not compound 1, or a pharmaceutically acceptable salt thereof. In various embodiments, the subject is human.

[0112] In various embodiments, the additional therapeutic agent is an antiviral agent. In another embodiment, the additional therapeutic agent is an immunomodulatory agent, such as an immunosuppressive agent. In some embodiments, the additional therapeutic agent is a compound listed in Table B.

[0113] In some embodiments, the additional therapeutic agent is a nucleoside reverse transcriptase and translocase inhibitor. In some embodiments, the additional therapeutic agent is islatravir, which is discussed in U. S. Publication No. 2005 / 0215512, published September 29, 2005, herein incorporated by reference in its entirety'. In some embodiments, the additional therapeutic agent is a compound disclosed in International Publication No. WO 2015 / 148746, such as any of the compounds of Examples 1-5 disclosed therein, which is herein incorporated by reference in its entirety'. In some embodiments, the additional therapeutic agent is a prodrug of islatravir, such as a compound disclosed in International Publication Nos. WO 2021 / 050956 or WO 2021 / 050961, each of which is herein incorporated by reference in its entirety.

[0114] In some embodiments, the additional therapeutic agent is an HIV capsid inhibitor. In some embodiments, the additional therapeutic agent is lenacapavir (Sunlenca®). The method ofaction of lenacapavir is discussed in US Publication No. 2018 / 0051005, published February 22, 2018, which is herein incorporated by reference in its entirety. In some embodiments, the additional therapeutic agent is GS-CA1 (see Vidal, et al. Long-acting capsid inhibitor protects macaques from repeat SHIV challenges. Nature 601, 612-616 (2022), herein incorporated by reference in its entirety).

[0115] In some embodiments, the additional therapeutic agent is a nucleoside reverse transcriptase inhibitor. In some embodiments, the additional therapeutic agent is ulonivirine. The method of action of ulonivirine is discussed in International Publication No. WO 2014 / 058747, published April 17, 2014, herein incorporated by reference in its entirety.

[0116] Accordingly, in one embodiment, the present invention provides methods for treating a viral infection in a subject, the method comprising administering to the subject: (i) at least one compound 1 (which may include two or more different compounds), or a pharmaceutically acceptable salt thereof, and (ii) at least one additional therapeutic agent, wherein the amounts administered are together effective to treat a viral infection.

[0117] When administering a combination therapy of the invention to a subject, therapeutic agents in the combination, or a pharmaceutical composition or compositions comprising therapeutic agents, may be administered in any order such as, for example, sequentially, concurrently, together, simultaneously and the like. The amounts of the various actives in such combination therapy may be different amounts (different dosage amounts) or same amounts (same dosage amounts). Thus, for non-limiting illustration purposes, each of compound 1 and an additional therapeutic agent may be present in fixed dosage amounts in a single dosage unit (e.g., a capsule or a tablet).

[0118] In some embodiments, any of the disclosed compounds or a pharmaceutically acceptable salt thereof, is administered via injection, and the additional therapeutic agent is further administered via injection, using one or more injection devices. In some embodiments, the one or more injection devices is or includes a syringe, which can be employed manually, or as part of a syringe-containing injection device such as an autoinjector. A wide variety of injection devices can be used, including, but not limited to, a handheld or wearable autoinjector, a handheld or wearable manual injector, an on-body injector, a syrette, a jet injector, or a pen injector, each of which can be reusable or disposable.

[0119] In one embodiment, the compound is administered during a time when the additional therapeutic agent(s) exert their prophylactic or therapeutic effect, or vice versa.

[0120] In another embodiment, the compound and the additional therapeutic agent(s) are administered in doses commonly employed when such agents are used as monotherapy for treating a viral infection.

[0121] In another embodiment, the compound and the additional therapeutic agent(s) are administered in doses lower than the doses commonly employed when such agents are used as monotherapy for treating a viral infection.

[0122] In still another embodiment, the compound and additional therapeutic agent(s) are administered in doses lower than the doses commonly employed when such agents are used as monotherapy for treating a viral infection.

[0123] In one embodiment, the compound and the additional therapeutic agent(s) are present in the same composition. In some embodiments, this composition is suitable for oral administration. In one embodiment, this composition is suitable for subcutaneous administration. In another embodiment, this composition is suitable for intramuscular administration.

[0124] Viral infections and virus-related disorders that may be treated using the combination therapy methods of the present invention include, but are not limited to, those listed above. In some embodiments, the viral infection is HIV infection. In some embodiments, the viral infection is AIDS.

[0125] The compound and the additional therapeutic agent(s) may act additively or synergistically. A synergistic combination may allow the use of lower dosages of one or more agents and / or less frequent administration of one or more agents of a combination therapy. A lower dosage or less frequent administration of one or more agents may lower toxicity of therapy without reducing the efficacy of therapy. In one embodiment, the administration of at least one compound and the additional therapeutic agent(s) may inhibit the resistance of a viral infection to these agents.

[0126] In certain embodiments, the disclosed compounds or pharmaceutically acceptable salts thereof are combined with at least one HIV long-acting therapy, such as a long-acting injectable. Examples of drugs that are being developed as long-acting regimens include cabotegravir, rilpivirine, lenacapavir, tenofovir implant, ulonivirine, raltegravir, and long-acting dolutegravir. In some embodiments, the disclosed compounds are combined with lenacapavir. In some embodiments, the disclosed compounds are combined with ulonivirine.

[0127] As noted above, the present invention is also directed to use of compound 1 with one or more anti -HIV agents. An '‘anti -HIV agent” is any agent which is directly or indirectly effective in the inhibition of HIV reverse transcriptase or another enzyme required for HIV replication or infection, the treatment or prophylaxis of HIV infection, and / or the treatment, prophylaxis ordelay in the onset or progression of AIDS or ARC. It is understood that an anti-HIV agent is effective in treating, or delaying the onset or progression of, HIV infection or AIDS and / or diseases or conditions arising therefrom or associated therewith. For example, the compound of this invention may be effectively administered, e.g., post-exposure, in combination with effective amounts of one or more anti-HIV agents selected from HIV antiviral agents, immunomodulators, anti-infectives, or vaccines useful for treating HIV infection or AIDS. Suitable HIV antivirals for use in combination with the compound of the invention include, for example, those listed in Table B as follows:Table B

[0128] Some of the drugs listed in the table are used in a salt form; e.g., abacavir sulfate, indinavir sulfate, atazanavir sulfate, nelfinavir mesylate.

[0129] In one embodiment, one or more anti-HIV drugs are selected from lenacapavir, GS-CA1, lamivudine, abacavir, ritonavir, darunavir, atazanavir, emtricitabine, tenofovir, rilpivirine, doravirine, islatravir and lopinavir.

[0130] In some embodiments, the compound 1 is used in combination with lenacapavir or GS-CA1.

[0131] In still another embodiment, the compound 1 is used in combination with atazanavir. In another embodiment, the compound 1 is used in combination with darunavir. In another embodiment, the compound 1 is used in combination with rilpivirine. In one embodiment, the compound 1 is used in combination with lamivudine and abacavir.

[0132] In another embodiment, the compound 1 is used in combination with islatravir. In another embodiment, the compound 1 is used in combination with emtricitabine and tenofovir. In still another embodiment, the compound 1 is used in combination with doravirine. In still another embodiment, the compound 1 is used in combination doravirine, lamivudine and tenofovir DF. In another embodiment, the compound 1 is used in combination with ritonavir and lopinavir. In one embodiment, the compound 1 is used in combination with abacavir and lamivudine. In another embodiment, the compound 1 is used in combination with lopinavir and ritonavir.

[0133] In one embodiment, the present invention provides pharmaceutical compositions comprising (i) compound 1 or a pharmaceutically acceptable salt thereof; (ii) a pharmaceutically acceptable carrier; and (iii) one or more additional anti-HIV agents selected from doravirine, islatravir, ulonivirine, lenacapavir, abacavir, lamivudine, ritonavir and lopinavir, or a pharmaceutically acceptable salt or prodrug thereof, wherein the amounts present of components (i) and (iii) are together effective for the treatment or prophylaxis of infection by HIV or for the treatment, prophylaxis, or delay in the onset or progression of AIDS in the subject in need thereof.

[0134] In another embodiment, the present invention provides a method for the treatment of infection by HIV, or the treatment or delay in the onset or progression of AIDS, in a subject inneed thereof, which comprises administering to the subject (i) compound 1 or a pharmaceutically acceptable salt thereof and (ii) one or more additional anti-HIV agents selected from doravirine, islatravir, ulonivirine, lenacapavir, abacavir, lamivudine, ritonavir and lopinavir, or a pharmaceutically acceptable salt or prodrug thereof, wherein the amounts administered of components (i) and (ii) are together effective for the treatment or prophylaxis of infection by HIV or for the treatment, prophylaxis, or delay in the onset or progression of AIDS in the subject in need thereof.

[0135] It is understood that the scope of combinations of the compound of this invention with anti-HIV agents is not limited to the agents listed in Table B but further includes in principle any combination with any pharmaceutical composition useful for the treatment or prophylaxis of AIDS. The HIV antiviral agents and other agents will typically be employed in these combinations in their conventional dosage ranges and regimens as reported in the art, including, for example, the dosages described in the Physicians' Desk Reference. Thomson PDR. Thomson PDR, 57thedition (2003), the 58thedition (2004), the 59thedition (2005), and the like. The dosage ranges for a compound of the invention in these combinations are the same as those set forth above.

[0136] The doses and dosage regimen of the other agents used in the combination therapies of the present invention for the treatment of HIV infection may be determined by the attending clinician, taking into consideration the approved doses and dosage regimen in the package insert; the age, sex and general health of the subject; and the type and severity of the viral infection or related disease or disorder. When administered in combination, the compound and the other agent(s) may be administered simultaneously (i.e.. in the same composition or in separate compositions one right after the other) or sequentially. This is particularly useful when the components of the combination are given on different dosing schedules, e.g., one component is administered once daily and another component is administered every six hours, or when the pharmaceutical compositions are different, e.g., one is a tablet and one is a capsule. A kit comprising the separate dosage forms is therefore advantageous.EXAMPLES

[0137] The following examples are meant to be illustrative and should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.General Methods

[0138] The following examples serve only to illustrate the invention and its practice. The examples are not to be construed as limitations on the scope or spirit of the invention. In these examples, all temperatures are degrees Celsius unless otherwise noted, and "room temperature" refers to a temperature in a range of from about 20 °C to about 25 °C. Reactions sensitive to moisture or air were performed under nitrogen using anhydrous solvents and reagents. The progress of reactions was determined by either analytical thin layer chromatography (TLC) performed with E. Merck-precoated TLC plates, silica gel 60F-254, layer thickness 0.25 mm or liquid chromatography with mass spectrometry (LC-MS). For HPLC / MS data, two HPLC conditions used were as follows: 1) LC1 (Shimadzu C18 Xtimate 3pm 2.1x30 mm column with gradient 10:90-80:20 v / v CH3CN / H2O + v 0.0375 % TFA over 0.9 min then hold at 80:20 v / v CH3CN / H2O + v 0.0375% TFA for 0.6 min; flow rate 1.2 mL / min, UV wavelength 220 & 254 nm); 2) LC2 (Agilent C18 Xtimate 3 pm 2.1 X 30 mm column with gradient 10:90-80:20 v / v CH3CN / H2O + v 0.0375 % TFA over 3.0 min then hold at 80:20 v / v CH3CN / H2O + v 0.0375 % TFA for 0.5 min; flow rate 0.8 mL / min, UV wavelength 220 & 254 nm). Mass analysis was performed with electrospray ionization in positive ion detection mode.1H NMR spectra were recorded on Varian or Bruker instruments at 400-500 MHz. Chemical shifts were reported in parts per million (ppm). Tetramethylsilane (TMS) was used as internal reference in CDCh solutions, and residual CH3OH peak or TMS was used as internal reference in CDsOD solutions. Coupling constants (J) were reported in hertz (Hz). Concentration of solutions was carried out on a rotary evaporator under reduced pressure or by lyophilization. Flash chromatography was performed on pre-packed silica gel columns using a commercial MPLC system.Abbreviations

[0139] For purposes of this disclosure, the following abbreviations have the indicated meanings:Intermediate Atert-butyl 4-(2-(5-(benzyloxy)-6-(methoxycarbonyl)-4-oxo-4 / 7-pyran-2-yl)ethyl)-2,2- dimethyloxazolidine-3-carboxylate

[0140] Intermediate A was prepared according to the synthetic procedure described in International Publication No. WO 2018 / 102485, which is incorporated by reference herein.EXAMPLE 1: Synthesis of compound 1(3a / ?.5. S')-A-(2.4-dif1uorobenz l)-2-eth l-5.8-dihydroxy-l.7-dioxo-2.3.3a.4.5.7-hexahydro-l / / - pyrazino[2,l,6-c< / |indolizine-6-carboxamideStep 1 - Synthesis of Intermediate Int-B,

[0141] To a solution of / e / 7-butyl 4-(2-(5-(benzyloxy)-6-(methoxycarbonyl)-4-oxo-477-pyran-2-yl)ethyl)-2,2-dimethyloxazolidine-3-carboxylate (3.8 g, 7.79 mmol) in 96 mL of DCM was added 24 mL of TFA. The reaction was stirred at RT for 40 min. The solvent was removed undervacuum to afford intermediate Int-B. This material was used in the next reaction without further purification. MS (ESI) m / z: 388.1 [M+H]+.Step 2 - Synthesis of Intermediate Int-C.

[0142] A solution of methyl 3-(benzyloxy)-6-(2-(2.2-dimethyloxazolidin-4-yl)ethyl)-4-oxo-4 / / -pyran-2-carboxylate (3.02 g. 7.80 mmol) in 140 mL of 2-propanol was heated at 85 °C for 1.5 h. It was cooled to RT. The solvent was removed under vacuum without heating. The residue was dissolved in 40 mL of EtOAc and then concentrated under vacuum to afford intermediate Int-C.This material was used in the next reaction without further purification. MS (ESI) m / z: 330.0 [M+H]+.Step 3 - Synthesis of Intermediate Int-D.

[0143] To a solution of methyl 6-(benzyloxy)-3-(hydroxymethyl)-7-oxo-1, 2,3,7-tetrahydroindolizine-5-carboxylate (860 mg, 2.61 mmol) in 30 mL of MeOH, was added ethanamine 2 M in THF (5.22 mL, 10.44 mmol). The reaction was stirred at RT overnight. The solvent was removed under vacuum. The residue was purified by a reverse phase C-18 column (275 g) eluting with 0-90% ACN (+ 0.05% TFA) / water (+ 0.05% TFA) gradient over 11 column) to give intermediate Int-D. MS (ESI) m / z: 343.4 [M+H]+.Step 4 - Synthesis of Intermediate Int-E,

[0144] To a solution of 6-(benzyloxy)-A-ethyl-3-(hydroxymethyl)-7-oxo-l, 2,3,7-tetrahydroindolizine-5-carboxamide (620 mg, 1.811 mmol, 1 eq.) in 7.5 mL of DCM stirred at 0 °C, was added A-ethyl-A-isopropylpropan-2-amine (0.959 mL, 5.43 mmol), followed by methanesulfonyl chloride (0.196 mL, 2.54 mmol). The reaction was stirred at 0 °C for 15 min. It was quenched by adding 2 eq. of MeOH. The resulting mixture was stirred at RT for 30 min. The solvent was removed under vacuum. The residue was purified by a silica gel column eluting with 5% MeOH / DCM to give intermediate Int-E. MS (ESI) m / z: 421.0 [M+H]+.Step 5 - Synthesis of Intermediate Int-F,

[0145] To a solution of (6-(benzyloxy)-5-(ethylcarbamoyl)-7-oxo-I,2,3,7-tetrahydroindolizin-3-yl)methyl methanesulfonate (920 mg, 1.750 mmol) in 15 mL of DMF, was added cesium carbonate (2281 mg, 7.00 mmol). The reaction was stirred at RT for 18 h. The reaction content was diluted with 4 mL of water, the resulting mixture was purified by a reverse phase C-18 column (275 g) eluting with 0-90% ACN (+ 0.05% TFA) / water (+ 0.05% TFA) in gradient over 11 column to afford intermediate Int-F. MS (ESI) m / z: 325.1 [M+H]+.Step 6 - Synthesis of Intermediate Int-G.

[0146] To a solution of 8-(benzyloxy)-2-ethyl-3,3a,4,5-tetrahydro-177-pyrazino[2,l,6-ct / Jindolizine-I,7(2 / 7)-dione (540 mg, 1.665 mmol) in 10 mL of DCM, was added NIS (375 mg.1.665 mmol). The reaction was stirred at RT for 30 min. The solvent was removed under vacuum. The residue was purified by a reverse phase C-18 column (275 g) eluting with 0-90% ACN (+ 0.05% TFA) / water (+ 0.05% TFA) gradient over 11 column to give intermediate Int-G as its TFA salt form. MS (ESI) m / z: 450.9 [M+H]+.Step 7 - Synthesis of Intermediate Int-H,

[0147] To a solution of 8-(benzyloxy)-2-ethyl-6-iodo-3,3a,4,5-tetrahydro-17 / -pyrazino[2,l,6-c<7|indolizine-l,7(277)-dione, TFA- (240 mg, 0.426 mmol) in THF (4 mL) at -78°C under a atmosphere of nitrogen, 1 M solution of LiHMDS in THF (1.704 mL, 1.704 mmol) was added dropwise. After it was stirred at -78°C for 10 min. a solution of 3-phenyl-2-(phenylsulfonyl)-l,2-oxaziridine (223 mg. 0.852 mmol) in 1 mL of THF was added dropwise at -78 °C. The reaction was warmed to RT and stirred for 10 min. The reaction mixture was quenched by addition of 1 mL of saturated aq. NH4CI solution and extracted with DCM (2 X 100 mL). The combined organics were dried over anhydrous Na2SO4 and then filtered. The filtrate was concentrated under vacuum. The residue was purified by a reverse phase C18 (100 g) column eluting with 0-100% ACN (+ 0.05% TFA) / water (+ 0.05% TFA) (gradient) to provide intermediate Int-H as its TFA salt form. MS (ESI) m / z: 467.1 [M+H]+.Step 8 - Synthesis of Intermediate Int-I1, Int-I2, Int-I3 and Int-I4.

[0148] To a solution of 8-(benzyloxy)-2-ethyl-5-hydroxy-6-iodo-3,3a,4,5-tetrahydro-177-pyrazino[2, L6-cti]indolizine-l,7(2 / / )-dione, TFA- (98 mg, 0.169 mmol), A-ethyl-A-isopropylpropan-2-amine (0.118 mL, 0.677 mmol) and (2,4-difluorophenyl)methanamine (36.3 mg, 0.254 mmol) in DMSO (2 mL) was added tetrakis(triphenylphosphine)palladium(0) (39.1 mg, 0.034 mmol). The reaction mixture was stirred at 90 °C for 1 h under an atmosphere of CO. The reaction mixture was cooled to room temperature and then purified by a reverse phase C18 column (100 g) eluting with 0-100% ACN (+ 0.05% TFA) / water (+ 0.05% TFA) to provide 8-(benzyloxy)-N-(2,4-difluorobenzyl)-2-ethyl-5-hydroxy-l,7-dioxo-2,3,3a,4,5,7-hexahydro-lH-pyrazino[2,l,6-cd]indolizine-6-carboxamide as a mixture of stereoisomers. This material was further separated by chiral SFC (Column: OD 21 X 250 mm; Condition: 40% MeOH (0.2 N NH4OH) / CCh; 100 bar; 254 nM; 35 °C) to afford Int-I1 (the first eluting stereoisomer, > 98% de, and > 98% ee, MS (ESI) m / z: 510.3 [M+H]+). Int-I2 (the second eluting stereoisomer, MS (ESI) m / z: 510.3 [M+H]+), Int-I3 (the third eluting stereoisomer, MS (ESI) m / z: 510.3 [M+H]+), and Int-I4 (the fouth eluting stereoisomer, MS (ESI) m / z: 510.3 [M+H]+).Step 9 - Synthesis of Compound 1

[0149] To a solution of intermediate Int-Ii (31 mg, 0.061 mmol) in DMF (1 mL) stirred at RT, lithium chloride (25.8 mg, 0.608 mmol) was added and the reaction mixture was heated at 90 °Cfor 2 h under a nitrogen atmosphere. The reaction was cooled to RT and was purified by a reverse phase C18 (50 g) column eluting with 0-100% ACN (+ 0.05% TFA) / water (+ 0.05% TFA) gradient to provide compound 1 (ee > 98%, de >98%). MS (ESI) m / z: 420.2 [M+H]+NMR (400 MHz, CDCh) 5: 10.78 (br s, 1H), 7.41 - 7.30 (m, 1H), 6.88 - 6.77 (m, 2H), 5.73 (d, J = 7.3 Hz. 1H), 4.89 - 4.76 (m. 1H), 4.70 - 4.58 (m, 2H), 3.80 - 3.60 (m, 3H), 3.56 (dt. J= 14.5, 7.2 Hz, 1H), 2.57 (dd, J= 13.8, 6.1 Hz, 1H), 2.20 (ddd, J= 13.7, 9.8, 7.5 Hz, 1H), 1.27 (t, J= 7.2 Hz, 3H).EXAMPLE 2: Synthesis of Isomeric compounds 2, 3 and 4

[0150] Starting from the appropriately subscript-numbered intermediate, the following compounds were prepared using a similar procedure as described in step 9 of Example 1.Int-I2Compound 2 Int-I3Compound 3 Int-I4Compound 4EXAMPLE 3: Alternative synthesis of Compound 1

[0151] The following procedure represents an exemplary alternative procedure for making compound 1.Step 1: (7?)-(5-oxopy rrolidin-2-y Ijmethy 14-methylbenzenesulfonate.

[0152] DCM (950 kg) was charged to the reaction vessel followed by (R)-5-(hydroxymethyl)pyrrolidin-2-one (70 kg, 608 mol) and tri ethylamine (70 kg). The reaction mixture was cooled to 0 °C followed by the addition of TsCl (124.5 kg) and DMAP (7.3 kg). The mixture was stirred for 1 h at 0 °C and subsequently warmed to 20 °C. The reaction mixture was stirred at 20 °C for 18 h. The reaction mixture was washed with water (358 kg), 0.5 % HC1 aqueous solution (348 kg), water (350 kg), and the organic layer was concentrated to 420 kg under vacuum while keeping the temperature below 40 °C. (?)-(5-oxopyrrolidin-2-yl)methyl 4-methylbenzenesulfonate (0.4 kg) was charged into the reaction mixture and subsequently warmed to 25 °C. The organic layer was concentrated to 420 kg while under vacuum and a temperature below 40 °C and while charging ^-heptane (138.0 kg). This process was repeated two more times. An additional amount of ^-heptane (210 kg) was charged to the reaction mixture, and the temperature of the mixture was adjusted to 25 °C. Subsequently, the reaction mixture was cooled to 0 °C over 4 h and stirred at this temperature for 7 h. The cooled reaction mixture was filtered, and the cake was dried under vacuum with nitrogen for 20 h at 45 °C to provide 154.0 kg of (7?)-(5-oxopyrrolidin-2-yl)methyl 4-methylbenzenesulfonate. MS (ESI) m / z: 270.1 [M+H]+.Step 2: -5-((bcnzyl(cthyl )amino)mcthyl )pyrrolidin-2-onc.

[0153] MeCN (1236 kg) was charged to a reaction vessel followed by (?)-(5-oxopyrrolidin-2-yljmethyl 4-methylbenzenesulfonate (153.4 kg) and JV-benzylethanamine (77.1 kg). The reaction mixture was stirred for 30 min at 25 °C, and CsF (159.8 kg) was subsequently added. The reaction mixture was heated to 80 °C, and stirred at that temperature for 112 h. The reaction mixture was cooled down to 20 °C, and subsequently filtered. The organic phase was concentrated to 460 kg while under vacuum and below a temperature of 45 °C. The concentrated mixture was warmed to 40 °C, and DCM (760 kg) was charged to the reaction mixture. Theorganic layer was washed with water (460 kg, twice), and the organic layer was concentrated back to 460 kg while under vacuum and below a temperature of 40 °C. IPA (616.4 kg) was charged, and the reaction mixture was concentrated back to 460 kg under vacuum and below a temperature of 40 °C. This IPA addition / concentration process was repeated one more time to afford (A)-5-((benzyl(ethyl)amino)methyl)pyrrolidin-2-one as a solution (825.5 kg). MS (ESI) m / z: 233.2 [M+H]+.Step 3: tert-butyl (J?)-ethyl((5-oxopyrrolidin-2-yl)methyl (carbamate.

[0154] 40.0 kg of (7?)-5-((benzyl(ethyl)amino)methyl)pyrrolidin-2-one (313 kg solution in IP A) and IPA (80 kg) were charged to a reaction vessel. The reaction mixture was cooled to 0 °C and di-tert-butyl dicarbonate (42 kg) was subsequently charged. The reaction mixture was subsequently warmed to 25 °C, and Pd(OH)2 (8 kg) was charged to the vessel. The reaction mixture was put under a positive pressure of nitrogen followed by hydrogen (45 psi). The reaction mixture was warmed to 45 °C, and stirred at this temperature for 18 h. The reaction mixture was subsequently cooled to ambient temperature and filtered through Celite. The cake was washed with IPA (80 kg, twice) and combined with the filtrate. Subsequent concentration of the reaction mixture to 80 kg was performed while keeping the reaction mixture below 45 °C. n-Heptane (243 kg) was added to the reaction mixture and n-butyl (R)-ethyl((5-oxopyrrolidin-2-yl)methyl)carbamate (80 g) was added. The mixture was stirred at 25 °C for 3 h, and subsequently concentrated to 240 kg under vacuum and below 45 °C. w-Heptane (240 kg, twice) was added and concentrated to 240 kg. The reaction mixture was cooled to 0 °C over 8 h, and stirred at that temperature for an additional 4 h. Filtration of the mixture was performed, and the wet cake was dried for 18 h below 30 °C to afford 38.6 kg of tert-butyl (R)-ethyl((5-oxopyrrolidin-2-yl)methyl)carbamate. MS (ESI) m / z: [M+H]+243.1.Step 4: tert-butyl (J?)-((l-benzyl-5-oxopyrrolidin-2-yl)methyl)(ethyl)carbamate.

[0155] tert-butyl (R)-ethyl((5-oxopyrrolidin-2-yl)methyl)carbamate (38.1 kg, 157 mol), THF (160 kg), benzy l bromide (28.2 kg), and potassium tert-butoxide (212 kg) were charged to thereaction vessel. The mixture was stirred for 2 h at 25 °C. Citric acid monohydrate (12 kg) was subsequently added followed by water (120 kg). The reaction mixture was concentrated to 190 kg under vacuum and below 40 °C. MTBE (78 kg) was charged, and the organic layer was collected. The organic layer was washed with water (120 kg) and concentrated to 114 kg under vacuum and below 40 °C. DCM (205 kg) was then charged to the organic layer, and the organic layer subsequently concentrated to 114 kg. An additional amount of DCM (280 kg) was added, and the mixture was subsequently concentrated to 114 kg again while under vacuum and below 40 °C to provide a solution of tert-butyl ( / ?)-(( l-benzyl-5-oxopyrrolidin-2-yl)methyl)(ethyl)carbamate (116 kg). MS (ESI) m / z: 333.2 [M+H]+.Step 5: methyl (7?.£’)-2-(l-benzyl-5-(((terr-butoxycarbonyl)(ethyl)amino)methyl)pyrrolidin-2-ylidene)-4-methoxy-3-oxobutanoate.

[0156] A solution of tert-butyl (R)-((1-benzyl-5-oxopyrrolidin-2-yl)methyl)(ethyl)carbamate in DCM (750 g SM), DCM (3.75 L), and 2,6-di -to7-butyl-4-methylpyridine (70 g, 341 mmol) were charged to the reaction vessel. The reaction mixture was cooled to -55 °C.Trifluoromethanesulfonic anhydride (828 g, 2935 mmol) was added to the reaction mixture followed by methyl 4-methoxy-3-oxobutanoate (989 g, 6767 mmol) and DCM (2.25 L). The reaction mixture was w armed to 0 °C follow ed by addition of DBU (1.133 kg, 7442 mmol). Subsequent cooling of the reaction mixture to -40 °C was performed, followed by stirring at this temperature for 0.5 h. The reaction mixture was then warmed to 25 °C and stirred at that temperature for 15 h. The organic layer was concentrated in vacuo followed by addition of MTBE (3.75 L). The organic layer was washed with 10% citric acid (3.75 L, twice), 5% NaHCCh (3.75 L), and water (3.75 L). The organic layer was subsequently concentrated in vacuo to afford methyl (A, E’)-2-(l-benzyl-5-(((rerEbutoxycarbonyl)(ethyl)amino)methyl)pyrrolidin-2-ylidene)-4-methoxy-3-oxobutanoate (624 g, 1.36 mol). (MS (ESI) m / z: 461.3 [M+H]+.Step 6: methyl (R,E)-2-(1-benzyl-5-((ethylamino)methyl)pyrrolidin-2-ylidene)-4-methoxy-3-oxobutanoate.

[0157] methyl (?, E)-2-(l-benzyl-5-(((tert-butoxycarbonyl)(ethyl)amino)methyl)pyrrolidin-2-ylidene)-4-methoxy-3-oxobutanoate (3.65 kg, 7.93 mol), DCM (23 kg), and TFA (15.4 kg) were added to a reaction vessel. The reaction mixture was stirred for 16 h at 20 °C. The reaction mixture was washed with a potassium phosphate solution (15.8 kg K₃PO₄·3H₂O and 43.8 kg water), and the organic layer was separated. The aqueous phase was extracted with DCM (19 kg). The organic layers were combined and MgSO₄ (3.7 kg) was added. The slurry was filtered, and the cake was washed with DCM (12 kg). The filtrate was concentrated to 10 L under vacuum and below 30 °C. w-Heptane (13 kg) was added, and the mixture was stirred at 25 °C for 2 h.Subsequent filtration was performed, and the wet cake was added to an additional vessel with toluene (37 kg). The mixture was stirred at 20 °C for 1 h followed by filtration. The wet cake was dried at 25 °C under vacuum for 18 h to afford 4.83 kg (TFA salt). MS (ESI) m / z: 360.4 [M+H]+.methyl (R,E)-2-(5-((ethylamino)methyl)pyrrolidin-2-ylidene)-4-methoxy-3-oxobutanoate.

[0158] IPA (230 kg), methyl (R,E)-2-(1-benzyl-5-((ethylamino)methyl)pyrrolidin-2-ylidene)-4-methoxy-3-oxobutanoate (31.3 kg), and Pd(OH)2 / C (4.7 kg) were charged to a reaction vessel. The reaction vessel was put under a positive pressure of nitrogen followed by hydrogen (45 psi). The reaction mixture was warmed to 50 °C and stirred for 20 h. The reaction mixture was subsequently cooled to 25 °C and filtered through Celite (10 kg). The cake was washed with IPA (20 kg) and concentrated under vacuum and below 40 °C to afford a solution of methyl (R,E)-2-(5-((ethylamino)methyl)pyrrolidin-2-ylidene)-4-methoxy-3-oxobutanoate. MS (ESI) m / z: 271.2 [M+H]+.methyl (R,E)-2-(5-((benzyl(ethyl)amino)methyl)pyrrolidin-2-ylidene)-4-methoxy-3-oxobutanoate.

[0159] Methyl (A, E)-2-(5-((ethylamino)methyl)pyrrolidin-2-ylidene)-4-methoxy-3-oxobutanoate (10.8 kg), THF (16.7 L), and sodium carbonate (2.61 kg) were added to the reaction mixture, and the reaction mixture w as subsequently heated to 40 °C. Benzy l bromide (2.74 kg) was added to the reaction mixture, and the reaction mixture was stirred at 40 °C for 18 h. The reaction mixture was filtered, and MTBE (16.7 L) was added to the filtrate. The filtrate was washed with 15 wt% NaCl (10 kg), and the organic layer was dried with MgSO₄ and concentrated in vacuo to afford methyl ( / . A’)-2-(5-((benzyl(ethyl)amino)methyl)pyrrolidin-2-ylidene)-4-methoxy-3-oxobutanoate as an oil (5.72 kg). MS (ESI) m / z: 361.1 [M+H]+.dimethyl (A’)-3-((benzyl(ethyl)amino)melhyl)-6-methoxy-7-oxo-1.2.3.7-tetrahvdroindolizine-5.8-dicarboxylate.

[0160] Methyl (J?, E)-2-(5-((benzyl(ethyl)amino)methyl)pyrrolidin-2-ylidene)-4-methoxy-3-oxobutanoate (4.52 kg) and DMF (15.9 kg) were charged to the reaction mixture, followed by cooling of the reaction mixture to 5 °C. Methyl 2-chloro-2-oxoacetate (1.14 kg with 2 L DMF) was added dropwise and stirred at 5 °C for 25 h. An additional amount of methyl 2-chloro-2-oxoacetate (286 g with 0.5 L DMF) was added dropwise to the reaction mixture, and the reaction mixture was stirred for an additional 2 h at 5 °C. The reaction mixture was subsequently warmed to 25 °C and stirred for 18 h. IP Ac (48 kg) was charged to the reaction vessel, and the reaction mixture was washed with 10% ammonium acetate in water (27 kg). The aqueous layer was extracted with IP Ac (24 kg). The combined organic layers were washed with ammonium acetate (10% in water) (27 kg, three times). The organic layer was concentrated under vacuum below 35 °C to afford a black oil (4.3 kg). MS (ESI) m / z: 429.1 [M+H]+.methyl (R)-2-ethyl-8-methoxy-1,7-dioxo-2,3,3a,4,5,7-hexahydro-1H-pyrazino[2,1,6-cd]indolizine-6-carboxylate.

[0161] Dimethyl (R)-3-((benzyl(ethyl)amino)methyl)-6-methoxy-7-oxo-l, 2,3,7-tetrahydroindolizine-5,8-dicarboxylate (335.58 g), MeOH (1.0 kg), and Pd(0H)2 / C (39 g) were charged to a reaction vessel. The reaction vessel was purged with argon three times, followed by pressurizing with hydrogen (50 psi) at 25 °C. Reaction mixture was stirred for 18 h under these conditions. Reaction mixture was subsequently filtered and concentrated to 520 g under vacuum and at 40 °C. The reaction mixture was cooled to 25 °C and stirred at this temperature for 3 h. IP Ac (1.3 L) was added dropwise at this temperature. Subsequent concentration to 260 g under vacuum at 40 °C was performed followed by an additional addition of IP Ac dropwise (1.7 L). The mixture was cooled to 20 °C and stirred for 20 h at that temperature followed by filtration. The cake was dried at 25 °C for 16 h to afford methyl (R)-2-ethyl-8-methoxy-1,7-dioxo-2,3,3a,4,5,7-hexahydro-1H-pyrazino[2,1,6-cd]indolizine-6-carboxylate (67 g). MS (ESI)(R)-N-(2,4-difluorobenzyl)-2-ethyl-8-methoxy-1,7-dioxo-2,3,3a,4,5,7-hexahydro-1H-pyrazino[2,1,6-cd]indolizine-6-carboxamide. methyl

[0162] (R)-2-ethyl-8-methoxy-1,7-dioxo-2,3,3a,4,5,7-hexahydro-1H-pyrazino[2,1,6-cd]indolizine-6-carboxylate (1.66 kg), toluene (7.5 L), and Al(O‘Pr)3 (510.2 g) were charged to a reaction vessel. The reaction mixture was heated to 50 °C and stirred at that temperature for 18 h. An additional amount of Al(O‘Pr)3 (509.8 g) was added to the reaction vessel, and the reaction mixture was stirred for an additional 96 h at 50 °C. The reaction mixture was subsequently cooled to 0 °C and 1 M H₂SO₄ (aq.) (7.5 L) was added to the chilled reaction mixture. Stirring at this temperature for 1.5 h was performed, followed by filtration. The cake was washed with water (3 L followed by 1.5 L). The cake was dried under vacuum at 45 °C for 18 h to afford (R)-N-(2,4-difluorobenzyl)-2-ethyl-8-methoxy-1,7-dioxo-2,3,3a,4,5,7-hexahydro-1H-pyrazino[2,1,6-cd]indolizine-6-carboxamide (2.15 kg). MS (ESI) m / z: 418.2 [M+H]+.(R)-N-(2,4-difluorobenzyl)-2-ethyl-8-methoxy-1,7-dioxo-2,3,3a,4,5,7-hexahydro-1H-pyrazino[2,1,6-cd]indolizine-6-carboxamide.

[0163] (7?)- / V-(2,4-difluorobenzyl)-2-ethyl-8-methoxy-l,7-dioxo-2,3,3a,4,5.7-hexahydro-lH- pyrazino[2,l,6-cd]indolizine-6-carboxamide (4.32 kg) was dissolved in DCM (20 kg) in a reaction vessel followed by concentration to 8.6 L. Heptane (10 kg) was subsequently added at 25 °C. The reaction mixture was stirred for 1 h at 25 °C followed by filtration. The wet cake was dried under vacuum at 45 °C for 18 h to afford (R)-N-(2,4-difluorobenzyl)-2-ethyl-8-methoxy-1,7-dioxo-2,3,3a,4,5,7-hexahydro-1H-pyrazino[2,1,6-cd]indolizine-6-carboxamide (4.1 kg) as a solid (2.15 kg). MS (ESI) m / z: 418.2 [M+H]+.(3aR,5S)-N-(2,4-difluorobenzyl)-2-ethyl-5-hydroxy-8-methoxy-1,7-dioxo-2,3,3a,4,5,7-hexahydro-1H-pyrazino[2,1,6-cd]indolizine-6-carboxamide.

[0164] (7?)-N-(2,4-difluorobenzyl)-2-ethyl-8-methoxy-l,7-dioxo-2,3,3a,4,5,7-hexahydro-lH- pyrazino[2,l,6-cd]indolizine-6-carboxamide (400 g) and DME (6000 mL) were added to a dried flask purged with nitrogen and subsequently cooled to -20 °C. LiHMDS (1.0 M solution in DME, 3547 mL) was added at -20 °C over 30 min and subsequently stirred at -20 °C. Cumyl hydroperoxide (219 g) was added dropwise to the reaction at -20 °C over 1 h, stirred for 1 h, and allowed to warm to 25 °C and stir at 25 °C overnight. After overnight stirring at 25 °C, an aqueous solution of NaHSO₃ (4000 mL) was added dropwise over 10 min to reaction and stirred for 0.5 h at 20 °C. The solution was extracted with EtOAc (3 X2 L), and the combined organic layers were washed with brine (3 X 2 L). The solution was dried over MgSO₄, filtered, and concentrated to afford an oil (800 mL). MTBE (7 L) was added over 5 min to the oil, and the mixture was stirred for 0.5 h. Heptane (7 L) was added over 30 min to this solution. After stirring overnight, this mixture was filtered to give 343.8 g of (3aR,5S)-N-(2,4-difluorobenzyl)-2-ethyl-5-hydroxy-8-methoxy-1,7-dioxo-2,3,3a,4,5,7-hexahydro-1H-pyrazino[2,1,6-cd]indolizine-6-carboxamide as a solid. MS (ESI) m / z: 434.1 [M+H]+.(J< / ,. TS')-A’-(2.4-difluorobenzyl)-2-ethyl-5.8-dihvdroxy-1.7-dioxo-2.3.3a.4.5.7-hexahvdro-lH-pyrazino[2.1.6-cd1indolizine-6-carboxamide.

[0165] (3a7?,5< S)-Ar-(2,4-difluorobenzyl)-2-ethyl-5-hydroxy-8-methoxy-l,7-dioxo-2,3,3a,4,5,7-hexahydro-lH-pyrazino[2.1.6-cd]indolizine-6-carboxamide (408.4 g) and MeCN (4.08 L) were added to a dried flask and cooled to 0 °C. MgBr₂ (460 g) was added in several portions over 20 min while the reaction mixture was stirred at 0 °C. The reaction mixture was subsequently warmed to 25 °C over 1 h and stirred for 2 h at this temperature. 5% H₂SO₄ (4.1 L) was added over 10 min to the reaction mixture with subsequent stirring at 20 °C. Extraction with EtOAc (4 X 3 L) was performed, and the combined organic layers were washed with water (3 X 3 L). The combined organic layers were then dried over MgSO₄ with filtration and concentration to 6.8 L. Activated carbon (52 g, 20 w / w%) was added to the concentrated mixture and stirred for 1 h at 50 °C with subsequent filtration. The activated carbon was washed with MeCN (650 mL) at 50 °C at 1 h followed by filtration. The combined organic layers were distilled to 1.3 L and cooled to 25 °C and stirred overnight. After overnight stirring, the mixture was filtered to afford 221.6 g of solids. This cake was re-dissolved in EtOAc: MeCN (3:1, 10L), and activated carbon (11.1 g, 5 w / w%) was subsequently added. The solution was distilled to 1.1 L with subsequent cooling to RT. Heptane (1.1 L) was added and the mixture was stirred overnight, followed by filtration to afford (3aR,5S)-N-(2,4-difluorobenzyl)-2-ethyl-5,8-dihydroxy-1,7-dioxo-2,3,3a,4,5,7-hexahydro-1H-pyrazino[2,1,6-cd]indolizine-6-carboxamide, i.e., compound 1 (212.9 g), as a solid. 'H NMR (500 MHz, CDC13) δ 11.51 (s, 1H), 10.74 (t, J = 5.5 Hz, 1H), 7.39 (q, J = 8.4 Hz, 1H), 6.90 - 6.80 (m, 2H), 5.62 (d, J= 12 Hz, 1H), 4.98 (s, 1H), 4.81 - 4.71 (m, 1H), 4.62 (ddd, J= 56.6, 15.2, 5.7 Hz. 2H), 3.87 (t, J= 12.3 Hz, 1H). 3.71 (dt, J= 12.2, 6.4 Hz, 2H), 3.56 (dq. J= 14.2.7.2 Hz, 1H), 2.50 (dd, J= 13.6, 6.2 Hz, 1H), 2.31 - 2.22 (m, 1H), 1.29 (t, J= 7.2 Hz, 3H). MS (ESI) m / z: 420.2 [M+H]+.EXAMPLE 4: Antiviral Potency

[0166] Antiviral potency of compounds was determined based on their ability to block HIV-1 replication in a GFP reporter cell line; this assay is referred to as VIKING. HIV-1 replication was monitored using MT4-gag-GFP clone D3 (hereafter designated as MT4-GFP) cells, which are MT4 cells modified to harbor a GFP reporter gene, the expression of which is dependent on theHIV-1 proteins tat and rev. MT4 is a human T-cell line that is particularly susceptible to HIV-1 infection. HIV-1 infection of MT4-GFP cells results in GFP expression approximately 24 h postinfection.

[0167] MT4-GFP cells were maintained at 37 °C I 5% CO2 / 90% relative humidity in RPMI 1640 supplemented with 10% fetal bovine serum. 100 U / mL penicillin / streptomycin. and 400 pg / mL geneticin (G418) to maintain the reporter gene. For infections, MT4-GFP cells were placed in the same medium lacking G418 and infected overnight with HIV-1 wild-type (R8) virus at an approximate multiplicity of infection of 0.01 in the same incubation conditions.

[0168] Cells were then washed and resuspended in RPMI 1640 containing 10% normal human serum at 1.6 × 103cells / mL. Compound plates were prepared by dispensing compounds dissolved in DMSO into wells of 384-well poly D lysine-coated plates (0.2 pl / well) using an ECHO acoustic dispenser. Each compound was tested in a 10-point serial 3-fold dilution (typical final concentrations: 40 pM - 2 nM). Controls included no inhibitor (DMSO only) and a combination of three antiviral agents as a positive control (efavirenz, indinavir, and an integrase strand transfer inhibitor at final concentrations of 4 pM each). Infected cells were added (50 pL / well) to compound plates and were maintained at 37° C / 5% CO2 / 90% relative humidity. Infected cells were quantified at two time points, approximately 48 h and 72 h post-infection, by counting the number of green cells in each well using an Acumen eX3 scanner. The increase in the number of green cells over ~24 h period gives the reproductive ratio, R, which is calculated by dividing the number of green cells at 72 h over those at 48 h post-infection. The percent inhibition caused by a test compound is calculated using the following formula:inhibition [l-(Rtestcompound Rpositivecontrol) / (RDMSOonly—Rpositivecontrol)] * 100*

[0169] The dose-response curves of each test compound were plotted as % inhibition versus the compound concentration. IC50 values were determined by non-linear 4-parameter fitting of the dose-response curves.

[0170] First, the isomeric mixture containing Compounds 1, 2, 3 and 4 was evaluated in the 10% NHS Viking assay and demonstrated an IC50 of 30.3 nM with wild-type (WT) HIV-1 virus and an IC50 of 1228 nM with the G140S / Q148H double mutant (Table 1). Subsequently, the antiviral activity of compound 1 was tested in a separate experiment, where it demonstrated an IC50 of 42.72 nM with WT HIV-1 and an IC50 of 121.8 nM with the G140S / Q148H double mutant HIV virus (Table 1).

[0171] As discussed above, Q148 is a dominant mutation to INSTI resistance, and G140S is a compensatory mutation that typically occurs after the appearance of Q148H. Q148H and G140Stogether are prevalent HIV integrase mutations in patients living with HIV that contribute to resistance to several INSTIs, including raltegravir and cabotegravir.

[0172] The data in Table 1 shows a 40.6-fold change in G140S / Q148H potency (compared to WT potency) for the isomeric mixture, but only a corresponding 2.9-fold change in double mutant potency for compound 1. This represents a 14-fold improvement in potency shift against the double mutant for compound 1. While potency values from biological assays ty pically come with some variability' from one experiment to another, the Viking assay is relatively robust and demonstrates a ty pical variance of <3-fold of a potency value. Therefore, the difference in mutant potency fold-changes between compound 1 and the isomeric mixture indicates a substantial disparity in the ability of the two molecules to inhibit G140S / Q148H compared to WT virus. Specifically, compound 1 demonstrates substantially greater efficacy of inhibiting replication in the G140S / Q148H variant compared to the isomeric mixture.Table 1. Potency values (nM) for Examples in the HIV-1 Viking assay with 10% normal human serum (NHS).EXAMPLE 5: Pharmacokinetic (PK) Studies of Compound 10.5% MC Formulations

[0173] Compound 1 was spray dried at 33% drug loading in HPMCAS-L grade from acetone yielding the intermediate batch of Compound 1 (amorphous phase). The spray dried intermediate of Compound 1 was vortexed / sonicated / stirred in 0.5% MC in water to achieve a suspension at a concentration of 20 mg / mL for 100 mg / kg administration to Wistar Hannover rats. Compound 1 was vortexed / sonicated / stirred in 0.5% MC in w ater to achieve a suspension at a concentration of 5 mg / mL for 25 mg / kg administration to beagle dogs. All suspensions were assumed to be homogenous. The dose formulations were stirred overnight prior to dosing.

[0174] Compound 1 was vortexed, sonicated, and stirred in 0.5% MC / 0.1% SLS in w ater to achieve a suspension at a concentration of 2 mg / mL for 10 mg / kg administration to beagle dogs and rhesus macaques, or 5 mg / mL for 25 mg / kg administration to beagle dogs. All suspensions were assumed to be homogenous. The 10 mg / kg formulations were dosed within 4 h of suspension preparation. The 25 mg / kg formulation was stirred overnight prior to dosing.

[0175] Compound 1 was vortexed, sonicated, and stirred in 0.5% MC / 0.25% SLS in water to achieve a suspension at a concentration of 1 mg / mL for 5 mg / kg administration to beagle dogs. The suspension was assumed to be homogenous. The bottle was stirred overnight prior to dosing, protected from light.

[0176] The spray dried intermediate of Compound 1 was vortexed / sonicated / stirred in 0.5% MC / 0.25% SLS / 5 mM HC1 in water to achieve a suspension at a concentration of 1 mg / mL for 5 mg / kg administration, a concentration of 3 mg / mL for 15 mg / kg administration, or a concentration of 6 mg / mL for 30 mg / kg administration to beagle dogs. All suspensions were assumed to be homogenous. Animals were dosed within 4 h of suspension preparation.DMSO: PEG400 (50:50, v / v) Formulations

[0177] Compound 1 was dissolved in a mixture of DMSO: PEG400 (50:50, v / v) to achieve a solution at a concentration of 0.25 mg / mL for 0.1 mg / kg, or a concentration of 0.5 mg / mL for 0.2 mg / kg, IV administration to Wistar Hannover rats and 1 mg / mL for 0.1 mg / kg administration to beagle dogs and rhesus macaques. Animals were dosed within 4 h of preparing the dosing solutions.PEG400 Formulations

[0178] Compound 1 was vortexed. sonicated, and stirred in PEG400 to achieve a suspension at a concentration of 50 mg / mL for 100 mg / kg administration to Wistar Hannover rats, or 15 mg / mL for 30 mg / kg administration to beagle dogs. All suspensions were assumed to be homogenous. Animals were dosed within 4 h of suspension preparation.10% Tween80 Formulations

[0179] P. O. Formulation: Compound 1 was vortexed / sonicated / stirred in 10% Tween 80 in water to achieve either a solution at a concentration of 0.1 mg / mL for 0.5 mg / kg administration or a concentration of 0.2 mg / mL for 1 mg / kg administration, or a suspension at a concentration of 20 mg / mL for 100 mg / kg administration to Wistar Hannover rats. Compound 1 was vortexed / sonicated / stirred in 10% Tween 80 in water to achieve a suspension at a concentration of 0.1 mg / mL for 0.5 mg / kg administration, 2 mg / mL for 10 mg / kg administration, or 6 mg / mL for 30 mg / kg administration to beagle dogs. All suspensions were assumed to be homogenous. Animals were dosed within 4 h of preparing the dosing formulation.Animal PK Studies

[0180] Animals: Animals (male Wistar Hannover rats (300 to 356 g), male beagle dogs (9.9 to 13.7 kg), and male rhesus macaques (9.2 to 10.9 kg) were obtained from an approved vendor (Charles River Laboratories. Marshall Bioresources, or NIRC / The Mannheimer Foundation).

[0181] Acclimation / Quarantine: Following arrival, animals were assessed as to their general health by a member of the veterinary staff or other authorized personnel. Animals were acclimated for at least 3 days before being placed on study.

[0182] Animal Husbandry: Wistar Hannover rats were individually housed during acclimation through duration of the study. Beagle dogs and rhesus macaques were pair housed in the colony. Rhesus macaques were placed in study chairs in the morning of dosing day and returned to colony at the end of the day. They were placed in bleed chairs for subsequent time points then immediately returned to colony. Dogs were placed in individual cages one day prior to the study and are returned to the colony after the 24 h time point. Subsequent time points were taken in their home cage. The animal room environment was controlled (target conditions: temperature 18 to 26°C., relative humidity 30 to 70%, 12 h artificial light and 12 h dark). Temperature and relative humidity were monitored daily.

[0183] Animal Cannulation: Wistar Hannover rats were cannulated, but beagle dogs and rhesus macaques were not.

[0184] Animals were fasted at least 12 h prior to the administration. All animals had access to Certified Rodent and non-Rodent Diet (LabDiet 5002 certified rodent diet, LabDiet 5007 certified canine diet with Pedigree chopped ground dinner with chicken canned food, or LabDiet 5048 certified primate diet) ad libitum either 2 or 4 h post dosing.

[0185] Filtered water was provided to the animals via Lixit or bottle ad libitum. There were no known contaminants in the diet or water that, at the levels of detection, were expected to interfere with the purpose, conduct or outcome of the study.1. Dose Formulation

[0186] IV Formulation: Solutions were prepared on the day of dosing according to the procedure described above. Animals were dosed within 4 h of solution preparation.

[0187] P. O. Formulation: Solutions or suspensions w ere prepared on the day of dosing according to the procedure described above. Animals were either dosed within 4 h of dose formulation preparation or following overnight stirring of prepared formulation prior to dosing.2. Dose Administration

[0188] The dose formulations were administered via IV injection or oral dosing following facility SOPs.3. Sample Collection

[0189] Approximately 0.25 mL blood was collected from the carotid artery vein at each time point for cannulated rats using an automated blood sampler with built in cooler set at 4°C. Allblood samples were collected into microcentrifuge tubes containing 0.004 mL of K2EDTA (0.5M) as anti-coagulant in the cooler and kept until processed for plasma.

[0190] Approximately 0.5 mL blood was collected from the jugular vein for at each time point for dogs. All blood samples were transferred into microcentrifuge tubes containing 0.004 mL of K3EDTA (0.5M) as anti-coagulant and placed on wet ice until processed for plasma.

[0191] Approximately 0.5 mL blood was collected from the saphenous vein on day of dosing and from femoral vein from 24 h onwards for rhesus macaques. All blood samples were transferred into microcentrifuge tubes containing 0.004 mL of K3EDTA (0.5M) as anti-coagulant and placed on wet ice until processed for plasma.4. Blood / Plasma Processing

[0192] Blood: Rat blood samples were processed by centrifugation at approximately 4 °C, 3000 x g-force for 2 min within 30 min of collection, except for the overnight collections, which were kept at 4°C until processing the following morning. The resulting plasma layer was removed and stored in polypropylene tubes, flash frozen over dry ice and kept at -70±10 °C until LC / MS / MS analysis.

[0193] Blood: Dog and rhesus macaques blood samples were processed by centrifugation at approximately 4 °C, 3000 x g-force for 2 min within 30 min of collection. The resulting plasma samples were removed and stored in polypropylene tubes, flash frozen over dry ice and kept at -70±10 °C until LC / MS / MS analysis.5. Sample AnalysisBioanalytical Method and Sample Analysis

[0194] LC-MS / MS methods for the quantitative determination of Compound 1 in plasma was developed under non-GLP conditions.

[0195] Calibration curves: Three calibration curves with 13 non-zero calibration standards were applied for the method including LLOQ.

[0196] The study sample analysis will be performed concurrently with 3 sets of calibration standards using the LC-MS / MS method.Acceptance Criteria

[0197] Linearity: a minimum of 6 calibration standards was back calculated to within ±25% of their nominal values in plasma. Specificity: The mean calculated concentration in the single blank matrix should be 0.33 times the LLOQ. Sensitivity: the LLOQ targeted 1 nM

[0198] Carryover: the mean calculated carry-over concentration in the single blank matrix sample analyzed immediately after the highest standard injection was set as the LLOQ. If thecarryover couldn't meet the criteria, then the percent of carryover should be estimated following in-house bioanalytical SOP.

[0199] The plasma was analyzed for Compound 1 using LC-MS / MS. All Compound 1 stocks were prepared in DMSO at 10 mM. Standards were prepared by using an HP D300 Digital Dispenser (HP. Palo Alto, CA). Calibration standards, and in vivo study plasma samples were processed by protein precipitation by addition of acetonitrile containing 0.1% formic acid with IS (labetalol, imipramine, and diclofenac) at 1:4 v / v ratio. Supernatant obtained from centrifugation (1811 x g-force) of precipitated plasma samples were directly injected for LC-MS / MS analysis.

[0200] Liquid chromatography was carried out on a Waters Transcend LX2 multiplexed UPLC system. Chromatographic separation was performed on a Waters XSELECT HSS T3 XP (2.1 mm x 50 mm x 2.5 pm) column maintained at ambient temperature with an injection volume of 5-10 pL. The mobile phase consisted of a solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) was delivered at a flow rate of 0.75 mL / min. The LC gradient started from 80 / 20% (A / B) and changed to 2 / 98% (A / B) from 0.25 to 1.75 min (ramp) and maintained this ramp for 0.42 min (step). The gradient was decreased to 80 / 20% (A / B) at 2.2 min (step) and maintained at this ratio for 0.83 min. Detection was carried out using a triple quadrupole tandem mass spectrometer (API4500 or API5000, AB Sciex, Foster City, CA) equipped with a turbo ion spray interface. Ions were created in the positive ion mode setting the sprayer voltage at 5.5 kV and the ion source temperature at 500 °C. The common parameters and the nitrogen flow values for nebulizer gas (Gas 1), auxiliary gas (Gas 2), curtain gas, and the gas for the CAD were set to 60, 40, and 9, respectively. The Analyst 1.7.2 software (AB Sciex) was used to control the MS-MS system and MultiQuant 3.0.3 for data analyses. Detection of Compound 1 was performed in the multiple reaction monitoring mode.6. Data Analysis

[0201] Pharmacokinetic parameters were obtained using non-compartmental methods (Watson LIMS® version 7.6.1, ThermoFisher Scientific, Waltham, MA). The AUCo-twere calculated from the first time point (0 min) up to the last time point with measurable drug concentration using the linear trapezoidal or linear log-linear trapezoidal rule. The remaining AUCt-inf was estimated by dividing the observed concentration at the last time point by the elimination rate constant (ke). This value was added to AUCo-t to estimate the AUCo-inf. The percent AUC extrapolated was a function of (AUCo-inf - AUCo-t) *100 / AUCo-inf.

[0202] The IV clearance value (CLP) was calculated by dividing the dose by AUCo-inf. The terminal ti / 2 was determined by unweighted linear regression analysis of the log transformeddata. The time points for determination of t1 / 2were selected by visual inspection of the data. The Vdss was calculated from the product of CLPand MRT.AUMCVdss= CLpx MRT, where MRT =AUC

[0203] The Cmax and the Tmax were obtained from the plasma concentration-time data. The percent absolute oral bioavailability (%F) was calculated as per the equation below. The absolute bioavailability (i.e., relative to a corresponding IV dose of Compound 1) was calculated from the areas under the curve of plots of plasma concentration of Compound 1 extrapolated to infinity.7. Results

[0204] Table 1 shows the mean PK of Compound 1 in Wistar Hannover rats. Compound 1 exhibited low CLp (0.008 to 0.009 mL / min / kg), a small Vdss (0. 12 to 0.15 L / kg), and a terminal ti / 2 of 193 to 236 h following IV administration in DMSO: PEG400 (50:50, v / v). Following P. O. administration at a dose of 0.5 mg / kg in 10% Tween80 in water, the Cmax of Compound 1 was reached approximately 3 h post-dose, and the average absolute bioavailability was 84%.Following P. O. administration at a dose of 1 mg / kg in 10% Tween80 in water, the Cmax of Compound 1 was reached approximately 4 h post-dose, and the average absolute bioavailability was 61%. Compound 1 was dosed as an oral suspension in 10% Tween80 in water, PEG400, or as the spray dried intermediate in 0.5% MC in water at a dose of 100 mg / kg. The absolute bioavailability was 20%, 22%, or 16%, respectively.Table 2: PK results in RatbData from studies represent mean values of n=2Studies were conducted in a non-crossover design

[0205] The plasma concentration versus time plots of Compound 1 in rats between intervals 0.03 h and 528 h after IV or P. O. administration, is shown in Figure 1. Compound 1 exhibited a mean MRT in rats of 270 to 325 h.

[0206] Table 3 shows the mean PK of Compound 1 in beagle dogs. Compound 1 exhibited low CLP(0.013 mL / min / kg), a small Vdss(0.12 L / kg), and a terminal t1 / 2 of 103 h following IV administration in DMSO: PEG400 (50:50, v / v). Following P. O. administration of Compound 1 at a dose of 0.5 mg / kg in 10% Tween80 in water, the Cmax was reached approximately 1 h postdose. and the average absolute bioavailability was 100%. Following P. O. administration of Compound 1 at a dose of 10 mg / kg or 30 mg / kg in 10% Tween80 in water, the absolute bioavailability was 81% and 31%, respectively. The Cmax increased less than dose proportionally with an 18-fold increase, compared to a 60-fold increase in dose. The bioavailability of Compound 1 was measured in a 0.5% MC suspension in the presence of SLS surfactant of varying strengths following P. O. administration at a dose of 5 mg / kg. The absolute bioavailability of Compound 1 was comparable dosed either as a suspension in 0.5% MC / 0.25% SLS in water or dosed as a suspension in 0.5% MC of the spray dried intermediate in the presence of 0.25% SLS / 5 mM HC1 in water. Specifically, the absolute bioavailabilities were observed to be 61 and 57%, respectively.

[0207] The absolute bioavailability of Compound 1 was 35% and 15%, respectively following a 15 mg / kg or 30 mg / kg dose of the spray dried intermediate in the presence of 0.25% SLS / 5 mM HC1 in water. Following P. O. administration of Compound 1 at doses of 10 and 25 mg / kg as a suspension in 0.5% MC / 0.1% SLS in water, the absolute bioavailability of Compound 1 was 19, and 11%, respectively. Following P. O. administration of the spray dried intermediate of Compound 1 at a dose of 25 mg / kg as a suspension in 0.5% MC, the absolute bioavailability of Compound 1 was 17%. Compound 1 was dosed as an oral suspension of the spray dried intermediate in 0.5% MC in the presence of 0.25% SLS / 5 mM HC1 in water at a dose of 30 mg / kg. The absolute bioavailability was 15%. An additional formulation of Compound 1 in PEG400 was tested in n=2 male beagle dogs at a dose of 30 mg / kg. This resulted in an observed bioavailability of 21%.Table 3: PK results in DogaData from studies represent mean values of n=3 ± standard deviationbData from studies represent mean values of n=2Studies were conducted in a non-crossover design

[0208] The plasma concentration versus time plots of Compound 1 in beagle dogs between intervals 0.03 h and 552 h after IV or P. O. administration, is shown in Figure 2. Compound 1 exhibited a mean MRT in dogs of 125 h (±36 h).

[0209] Table 4 shows the mean PK of Compound 1 in rhesus macaques. Compound 1 exhibited low CLP(0.009 mL / min / kg). a small Vdss (0.12 L / kg), and a terminal t1 / 2 of 185 h following IV administration in in DMSO: PEG400 (50:50, v / v). Following P. O. administration of Compound 1 at a dose of 10 mg / kg in 0.5% MC / 0.1% SLS in water, the Cmax was reached approximately 12 h post-dose, and the average absolute bioavailability was 34%.

[0210] The plasma concentration versus time plot of Compound 1 concentration over time in rhesus macaques between intervals 0.03 h and 576 h after IV or P. O. administration, is shown in Figure 3. Compound 1 exhibited a mean MRT in rhesus of 225 h (±37 h).Table 4: PK results in Rhesus_ _IntravenousData from all studies represent mean values of n=3 ± standard deviationStudies were conducted in a non-crossover design

[0211] Based on this data, administration of compound 1 (at a dose of 0.1 mg / kg) provided half-life values as high as 103, 185, and 193 hours in dogs, macaques, and rats, respectively. Furthermore, this administration provided clearance values as low as 0.013, 0.009, and 0.008 mL / min / kg in dogs, macaques, and rats, respectively. These values represent substantially higher half-life and lower clearance values than those expected for the corresponding administration of compound 2, compound 3 or compound 4.

[0212] Generally, the CLP, Vdss, and AUCo-infPK values observed following IV administration correlate to corresponding PK parameters following oral administration. Half-life is a secondary PK parameter, as its value is dependent on the CLand Vdss values. The particular CLP, Vdss, and AUCo-infvalues observed following IV administration of compound 1 to, e g., dogs and macaques, can be allometrically scaled to determine the corresponding CLP, Vdss, and AUCo-infvalues in humans. Thus, the half-life values observed in, e.g., dogs and macaques, can be allometrically scaled to predict the corresponding human half-life following oral administration of compound 1.

[0213] Oral administration of 10 mg / kg of compound 1 in a 0.5% methylcellulose suspension provided an F value of 34% in macaques, and oral administration of 5 mg / kg in a 0.5% methylcellulose suspension provided an F value of 61% in dogs.

[0214] Overall, the PK properties observed in mammals as described Tables 1-3, combined with in vitro potency data (see Table 1) indicate that compound 1 is suitable for once-weekly oral dosing to subjects in need thereof (e g., human subjects).

[0215] The disclosed subject matter is not to be limited in scope by the specific embodiments and examples described herein. Indeed, various modifications of the disclosure in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0216] All references (e.g.. publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.

Claims

What is claimed is:

1. A compound of formula 1:or a pharmaceutically acceptable salt thereof.

2. A composition comprising compound 1, wherein the compound is present in an isomeric excess of at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 98%, at least 99%, or at least 99.5%.

3. The composition of claim 2, wherein the compound is present in an enantiomeric excess of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 98%, or at least 99%. or at least 99.5%.

4. The composition of claim 2 or 3, wherein the composition is substantially free of other isomers of the compound.

5. A pharmaceutical composition comprising an effective amount of the compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 5, wherein the compound is present as a crystalline solid.

7. The pharmaceutical composition of claim 5 or 6, wherein the composition is adapted for oral administration.

8. The pharmaceutical composition of any of claims 5-7, wherein the composition is adapted for once-weekly oral administration.

9. The pharmaceutical composition of any of claims 5-8, wherein the composition is present in a tablet or capsule.

10. The pharmaceutical composition of any of claims 5-9, wherein the composition provides a mean plasma half-life of at least 90 hours, at least 100 hours, at least 125 hours, at least 150 hours, at least 175 hours, at least 200 hours, or at least 225 hours, following administration to a mammalian subject.

11. The pharmaceutical composition of any of claims 5-10, wherein the composition is present in a suspension.

12. The pharmaceutical composition of claim 11, wherein the suspension comprises methylcellulose and sodium lauryl sulfate.

13. A method for the inhibition of HIV integrase in a subject in need thereof which comprises administering to the subject an effective amount of the compound of claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of claims 5-12.

14. A method for the treatment of infection by HIV or for the treatment or delay in the onset or progression of AIDS in a subject in need thereof, which comprises administering to the subject an effective amount of the compound of claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of claims 5-12.

15. The method of claim 13 or 14, wherein the compound, or a pharmaceutically acceptable salt thereof, is administered to the subject once weekly.

16. The method of any one of claims 13-15, wherein the compound, or a pharmaceutically acceptable salt thereof, is administered to the subject in one or more tablets.

17. The method of any one of claims 13-16 further comprising administering to the subject one or more additional therapeutic agents selected from doravirine, islatravir, ulonivirine, lenacapavir, abacavir, lamivudine, ritonavir and lopinavir, wherein the amounts administered ofthe compound and the one or more additional therapeutic agents, are together effective to treat infection by HIV or to treat, prevent or delay the onset or progression of AIDS.

18. The method of any one of claims 13-17, wherein the subject is human.

19. A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, for use in the preparation of a medicament for the inhibition of HIV integrase, for the treatment of infection by HIV, or for the treatment or delay in the onset or progression of AIDS in a subject in need thereof.

20. A tablet comprising an effective amount of compound 1, and a pharmaceutically acceptable carrier, wherein the tablet is adapted for once-weekly oral administration.

21. A method of reducing the risk for development of antiviral treatment resistance due to an HIV integrase mutation in a human subject infected with HIV, comprising administering an effective amount of the compound of claim 1, or the pharmaceutical composition of any one of claims 5-12.

22. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, for use in therapy.

23. The compound of claim 22, wherein the use comprises once-weekly administration.

24. A method of making compound 1, or a pharmaceutically acceptable salt thereof, comprising a step of iodinating 8-(benzyloxy)-2-ethyl-3,3a,4,5-tetrahydro-1H-pyrazino[2,1,6-cd]indolizine-1,7(2H)-dione, to generate 8-(benzyloxy)-2-ethyl-6-iodo-3.3a,4,5-tetrahydro-l / 7-pyrazino[2,l,6-c< Z]indolizine-I,7(2T0-dione.

25. The method of claim 24 further comprising a step of installing a hydroxy group onto the 8-(benzydoxy)-2-ethyl-6-iodo-3,3a,4,5-tetrahydro-I7f-pyrazino[2, I,6-c< |indohzine-I,7(2 / 7)-dione.

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